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Methods in Pharmacology
and Toxicology

Kevin R. Ward
Paul Matejtschuk Editors

Lyophilization
of Pharmaceuticals
and Biologicals
New Technologies
and Approaches
METHODS IN PHARMACOLOGY AND
TOXICOLOGY

Series Editor
Y. James Kang
Department of Pharmacology & Toxicology
University of Louisville, Louisville
Kentucky, USA

For further volumes:

http://www.springer.com/series/7653
Lyophilization of Pharmaceuticals
and Biologicals

New Technologies and Approaches

Edited by

Kevin R. Ward
Biopharma Process Systems Ltd., Winchester, UK

Paul Matejtschuk
National Institute for Biological Standards & Control (NIBSC), Potters Bar, Hertfordshire, UK
Editors
Kevin R. Ward Paul Matejtschuk
Biopharma Process Systems Ltd. National Institute for Biological Standards & Control
Winchester, UK (NIBSC)
Potters Bar, Hertfordshire, UK

ISSN 1557-2153 ISSN 1940-6053 (electronic)

Methods in Pharmacology and Toxicology
ISBN 978-1-4939-8927-0 ISBN 978-1-4939-8928-7 (eBook)
https://doi.org/10.1007/978-1-4939-8928-7
Library of Congress Control Number: 2018962406

© Springer Science+Business Media, LLC, part of Springer Nature 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
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The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
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This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of
Springer Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Dedication

This book is dedicated to the memories of Alan P. MacKenzie and Michael J. Pikal, who
contributed so much to the world of lyophilization.

v
Preface

So why another book on freeze-drying?

Lyophilization (freeze-drying) has been employed since the early twentieth century in
the medical field. While the principles of the process and the laws of physics governing it
remain unchanged, advances in technology and changes in the regulatory framework have
meant that the equipment has continued to evolve. Approaches to the development and
manufacturing processes have undergone a paradigm shift from a largely empirical trial-and-
error approach to one based upon initial determination of intrinsic physicochemical proper-
ties, followed by a more methodical approach to both formulation, process development
and monitoring. As editors who have between them over fifty years of working experience in
this field, we have witnessed these changes firsthand and so are delighted to be able to bring
together insightful contributions from many who are also actively involved in delivering
freeze-drying solutions at both laboratory and industrial scales.
The stabilization of active materials is a fundamental requirement across a range of
products, not limited to small drug molecules and biological materials, but also veterinary
products, advanced therapy medicines, and molecular diagnostics. Recent trends in the
pharmaceutical market alone have seen the rise to prominence of biopharmaceutical thera-
peutics [1] and the majority of these are lyophilized to ensure adequate stability and
competitive shelf life. Similarly, the rise in the use of molecular diagnostics and the power
of technologies such as polymerase chain reaction (PCR) assays mean that many of the
essential reagents contained within these assays kits must be lyophilized to ensure ease of
delivery.
There have been numerous texts and review articles devoted to the principles and
practicalities of lyophilization in the last 70 years, so this volume is not designed to rehash
what has previously been documented perfectly adequately elsewhere [2–5], nor indeed to
cover one specific aspect of lyophilization in detail, but rather, it seeks to highlight areas of
recent developments and technological advances in the field. In the present work, we have
drawn together experts in the particular facets of the lyophilization challenge in order to
update the reader with latest trends in each aspect. Advances in technology and improve-
ments in the way data are handled and presented have contributed to where we are today.
Developing freeze-drying knowledge in-house and purchasing one’s own equipment rather
than relying solely on external organizations or consultants are now the order of the day.
From providing initially an outline of the principles of effective product formulation and
analytical characterization of the physicochemical properties of the materials to be dried, we
progress to the discussion of one of the most widely impacting new developments in
industrial freeze-drying—that of controlled ice nucleation. Across two chapters, different
authors present and evaluate the full range of potential solutions, both those already
commercialized technologies and also the more developmental approaches.
Possibly one of the greatest changes we have witnessed in our careers is the move from a
trial-and-error-based approach to one that relies on prior understanding of the physico-
chemical aspects of a formulation before attempting to load the freeze-dryer. While some
analytical technologies had been developed prior to the twenty-first century [6, 7], they
were not necessarily widely applied in lyophilization due to the lack of understanding of the

vii
viii Preface

methods, the data interpretation, and how it could be applied in a real-life freeze-drying
cycle, especially in the days when chamber pressure and shelf temperature were themselves
not necessarily controlled or measured accurately.
The regulatory drivers have encouraged the increasing adoption of risk-based
approaches such as Quality by Design. This is addressed in this volume in a seminal
contribution from both former and present regulatory experts. These regulatory and
technological factors combine to present us with fresh challenges and new opportunities
in delivering freeze-drying solutions to stabilization problems. The principles and technol-
ogies that have developed as a result of these drivers have application beyond the narrow
specialty of pharmaceutical parenteral delivery and impact the development and manufac-
ture of in vitro diagnostics, novel therapeutics, and new-format vaccines right through to
nutraceuticals and foodstuffs.
Process Analytical Technology has become mainstream in the pharma industry and so its
presence is also increasingly felt within pharmaceutical freeze-drying. Three chapters are
therefore devoted to descriptions of both established and emerging technologies and how
they can be used practically within process development and the all-important scale up.
Developments in associated areas, such as the vital role of primary packaging, contain-
ment, and automation of industrial lyophilization, are also presented.
The demand for new high-dose therapeutic biologicals has led to specific challenges in
terms of both processing and freeze-drying of such drugs. These are reviewed in a dedicated
chapter on the aspects of delivering high concentration biotherapeutics at industrial scale.
The quality attributes of lyophilized products have also experienced a marked paradigm
shift over the past decade. Critical Quality Attributes are key to developing and consistently
manufacturing a licensed therapeutic and so with increasing pressure to define attributes
quantitatively rather than qualitatively, new technologies are emerging. The appearance and
structure of lyophiles is one such area, with time-consuming and subjective visual inspection
being supplanted by precise metrics such as cake resistance and robustness and the advent of
100% inspection of such properties by increasingly automated methodologies.
In this volume, we have aimed to bring together leading practitioners in the freeze-
drying community to address recent progress, not only in new analytical tools and applica-
tion of the data derived in cycle design but also in the manufacturing of lyophilized products
in the healthcare sector—whether these be therapeutics, vaccines, or diagnostic products—
and indeed the equipment to deliver this scale of freeze-drying.
Some of the contributions in this volume and the areas they cover are highlighted in the
following sections:
Analytical and Formulation Issues:
l New techniques for determination of critical formulation physicochemical parameters
and using multiple methods rather than a single one to arrive at consensus values—
Ward and Matejtschuk (Chap. 1)
l Steps to address subjectivity in interpretation of freeze-drying microscopy results—
Ward and Matejtschuk (Chap. 1)
l Traditional and Design of Experiment (DoE) approaches to formulation of complex
products are covered by Matejtschuk et al. (Chap. 2)
l Application of noninvasive testing methods for integrity of containers and moisture
content—Matejtschuk et al. (Chap. 2)
 Preface ix

l Challenges of understanding the interaction of multicomponent formulations and

strategies for handling complex biological samples (e.g., viruses)—Matejtschuk et al.
(Chap. 2)
Process Monitoring and Control:
l Review and assessment of Controlled Nucleation (CN) methods (technological
advances) are covered by Luoma et al. (Chap. 3) and Pisano (Chap. 4), illustrating
the variety of methods and the different underlying principles, status of commerciali-
zation and scalability, and impact on quality and consistency
l Use of specific Process Analytical Technology (PAT) equipment and methods is
described in detail by Kessler and Gong (Chap. 5) and Smith and Polygalov
(Chap. 11), while the role of statistical data in process understanding and scale-up is
illustrated by Bourlès et al. (Chap. 10)
l Regulatory aspects of freeze-drying, and the application of a Quality by Design
approach in this context, are covered by Awotwe-Atoo and Khan (Chap. 8)
l Understanding the impact of primary packaging (glass, closures, etc.) on the dried
product—still fundamental to long-term storage—is portrayed by McAndrew et al.
(Chap. 9)
l The need to control and contain the product during processing forms the basis of the
contribution by Cherry (Chap. 6) who also presents a novel approach to primary
packaging, while the automated handling technologies often used at large-scale man-
ufacture are highlighted by Guttzeit et al. (Chap. 7)
l The special challenges posed by highly concentrated protein products and their impact
on filling and lyophilization are discussed by Garidel and Presser (Chap. 12)
Post-lyophilization Analysis:
l Analysis of the structural and mechanical properties of lyophilized products and an
emerging technique to assess the Young’s modulus and strength of freeze-dried
materials are described by Hedberg et al. (Chap. 13)
l Solid-state Hydrogen-Deuterium Exchange Mass Spectrometry and molecular label-
ing are novel and powerful technologies for the detailed analysis of freeze-dried
proteins, the impact on structure, and their interactions with excipients—this is
presented by Balakrishna Chandrababu et al. (Chap. 14).
Inevitably, this is a snapshot only of the areas of progress within the field of lyophiliza-
tion toward the end of the second decade of the twenty-first century, and doubtless it will
have been to some degree influenced by the interests of the editors, both of whom come to
the field from a developmental perspective. However, as we have highlighted above, this area
has perhaps been the one which has developed most rapidly over the past decade. Also, it is
the rising clinical importance of these lyophilized materials that has driven the demand for
better and more robust process and product characterization.
We are indebted to all of the authors for their excellent contributions to this work and
recommend it to those seeking an update in this rapidly changing field. Finally, we thank
David C. Casey and all the team at Springer for their enthusiasm for the work and guidance
during its compilation.
x Preface

We hope the readers will share our enthusiasm for these topics as they are expounded by
the respective authors and find inspiration and encouragement to pursue their own interests
in the field of freeze-drying.

Winchester, UK Kevin R. Ward

Potters Bar, Hertfordshire, UK Paul Matejtschuk

References

1. Seymour P, Ecker DM (2017) Global biomanufacturing trends, capacity, and technology drivers:
industry biomanufacturing capacity overview. Am Pharmaceut Rev 20(4). Accessed from https://
dialog.proquest.com/professional/docview/1920294549
2. Hua T-C, Liu B-L, Zhang H (2010) Freeze-drying of pharmaceutical and food products. CRC Press/
Woodhead Publishing Ltd, Oxford
3. Haseley P, Oetjen G-W (2018) Freeze drying, 3rd edn. Wiley-VCH, Weinheim
4. Franks F, Auffret A (2008) Freeze drying of pharmaceuticals & biopharmaceuticals. Royal Society of
Chemistry, Cambridge
5. Rey L, May JC (eds) (2010) Freeze drying/lyophilization of pharmaceutical and biological products,
3rd edn. Informa, New York, NY
6. Rey L (2010) Glimpses into the realm of freeze drying: classical issues and new ventures. In: Rey L, May
JC (eds) Freeze-drying/lyophilization of pharmaceutical and biological products, 3rd edn. Informa,
New York, NY, pp 1–28
7. Ward K, Matejtschuk P (2010) The use of microscopy, thermal analysis and impedance measurements to
establish critical formulation parameters for freeze-drying cycle development. In: Rey L, May J (eds)
Freeze-drying/lyophilization of pharmaceuticals and biological products, 3rd edn. Informa Healthcare,
New York, NY; London, pp 112–135
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Characterization of Formulations for Freeze-Drying . . . . . . . . . . . . . . . . . . . . . . . . 1
Kevin R. Ward and Paul Matejtschuk
2 Formulation and Process Development for Lyophilized Biological
Reference Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Paul Matejtschuk, Kiran Malik, and Chinwe Duru
3 Controlled Ice Nucleation Using ControLyo®
Pressurization-Depressurization Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Jacob Luoma, Graham Magill, Lokesh Kumar, and Zakaria Yusoff
4 Alternative Methods of Controlling Nucleation in Freeze Drying . . . . . . . . . . . . . 79
Roberto Pisano
5 Tunable Diode Laser Absorption Spectroscopy in Lyophilization . . . . . . . . . . . . . 113
William J. Kessler and Emily Gong
6 Containment Options for the Freeze-Drying of Biological Entities
and Potent Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Chris Cherry
7 Freeze-Drying Systems: Freeze Dryer Interface Design Requirements
and Automatic Loading and Unloading Systems (ALUS™) . . . . . . . . . . . . . . . . . . 157
Maik Guttzeit, Carolin Wolf, Johannes Selch, and Thomas Beutler
8 Regulatory Aspects of Freeze-Drying. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
David Awotwe-Otoo and Mansoor Khan
9 Container and Reconstitution Systems for Lyophilized Drug Products . . . . . . . . 193
T. Page McAndrew, Douglas Hostetler, and Frances L. DeGrazio
10 Scale-Up of Freeze-Drying Cycles, the Use of Process
Analytical Technology (PAT), and Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . 215
Erwan Bourlès, Gael de Lannoy, Bernadette Scutellà,
Fernanda Fonseca, Ioan Cristian Trelea, and Stephanie Passot
11 Through Vial Impedance Spectroscopy (TVIS): A Novel Approach
to Process Understanding for Freeze-Drying Cycle Development. . . . . . . . . . . . . 241
Geoff Smith and Evgeny Polygalov
12 Lyophilization of High-Concentration Protein Formulations . . . . . . . . . . . . . . . . 291
Patrick Garidel and Ingo Presser
13 Mechanical Behavior and Structure of Freeze-Dried Cakes . . . . . . . . . . . . . . . . . . . 327
Sarah H. M. Hedberg, Sharmila Devi, Arnold Duralliu,
and Daryl R. Williams

xi
xii Contents

14 High-Resolution Mass Spectrometric Methods for Proteins

in Lyophilized Solids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Karthik Balakrishna Chandrababu, Rajashekar Kammari,
Yuan Chen, and Elizabeth M. Topp

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Contributors

DAVID AWOTWE-OTOO  Division of Post Marketing Activities II, Office of Pharmaceutical

Quality, Center for Drug Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, USA
KARTHIK BALAKRISHNA CHANDRABABU  Department of Industrial and Physical Pharmacy,
Purdue University, West Lafayette, IN, USA
THOMAS BEUTLER  GEA Lyophil GmbH, Hürth, North Rhine-Westphalia, Germany
ERWAN BOURLÈS  GSK Vaccines, Rixensart, Belgium
YUAN CHEN  Department of Industrial and Physical Pharmacy, Purdue University, West
Lafayette, IN, USA
CHRIS CHERRY  PDR, Cardiff Met University, Cardiff, UK
GAEL DE LANNOY  GSK Vaccines, Rixensart, Belgium
FRANCES L. DEGRAZIO  West Pharmaceutical Services, Inc., Exton, PA, USA
SHARMILA DEVI  Surfaces and Particle Engineering Group, Department of Chemical
Engineering, Imperial College London, London, UK
ARNOLD DURALLIU  Surfaces and Particle Engineering Group, Department of Chemical
Engineering, Imperial College London, London, UK
CHINWE DURU  Standardisation Science, National Institute for Biological Standards and
Control (NIBSC), Potters Bar, Herts, UK
FERNANDA FONSECA  UMR GMPA, AgroParisTech, INRA, Université Paris Saclay,
Thiverval-Grignon, France
PATRICK GARIDEL  Corporate Division Biopharmaceuticals, Process Science, Protein Science,
Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
EMILY GONG  Physical Sciences Inc., Andover, MA, USA
MAIK GUTTZEIT  GEA Lyophil GmbH, Hürth, North Rhine-Westphalia, Germany
SARAH H. M. HEDBERG  Surfaces and Particle Engineering Group, Department of Chemical
Engineering, Imperial College London, London, UK
DOUGLAS HOSTETLER  West Pharmaceutical Services, Inc., Exton, PA, USA
RAJASHEKAR KAMMARI  Department of Industrial and Physical Pharmacy, Purdue
University, West Lafayette, IN, USA
WILLIAM J. KESSLER  Physical Sciences Inc., Andover, MA, USA
MANSOOR KHAN  Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science
Center, College Station, TX, USA
LOKESH KUMAR  Genentech, Inc., South San Francisco, CA, USA
JACOB LUOMA  Genentech, Inc., South San Francisco, CA, USA
GRAHAM MAGILL  Genentech, Inc., South San Francisco, CA, USA
KIRAN MALIK  Standardisation Science, National Institute for Biological Standards and
Control (NIBSC), Potters Bar, Herts, UK
PAUL MATEJTSCHUK  National Institute for Biological Standards and Control (NIBSC),
Potters Bar, Hertfordshire, UK
T. PAGE MCANDREW  Scientific Communications, Scientific Affairs and Technical Services,
West Pharmaceutical Services, Inc., Exton, PA, USA
STEPHANIE PASSOT  UMR GMPA, AgroParisTech, INRA, Université Paris Saclay,
Thiverval-Grignon, France

xiii
xiv Contributors

ROBERTO PISANO  Department of Applied Science and Technology, Molecular Engineering

Laboratory, Politeccnico di Torino, Torino, Italy
EVGENY POLYGALOV  Pharmaceutical Technologies, Leicester School of Pharmacy,
De Montfort University, Leicester, UK
INGO PRESSER  Corporate Division Biopharmaceuticals, Process Science, Protein Science,
Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany
BERNADETTE SCUTELLÀ  GSK Vaccines, Rixensart, Belgium
JOHANNES SELCH  GEA Lyophil GmbH, Hürth, North Rhine-Westphalia, Germany
GEOFF SMITH  Pharmaceutical Technologies, Leicester School of Pharmacy, De Montfort
University, Leicester, UK
ELIZABETH M. TOPP  Department of Industrial and Physical Pharmacy, Purdue University,
West Lafayette, IN, USA
IOAN CRISTIAN TRELEA  UMR GMPA, AgroParisTech, INRA, Université Paris Saclay,
Thiverval-Grignon, France
KEVIN R. WARD  Biopharma Process Systems Ltd, Winchester, UK
DARYL R. WILLIAMS  Surfaces and Particle Engineering Group, Department of Chemical
Engineering, Imperial College London, London, UK
CAROLIN WOLF  GEA Lyophil GmbH, Hürth, North Rhine-Westphalia, Germany
ZAKARIA YUSOFF  SP Scientific, Warminster, PA, USA
 Chapter 1

Characterization of Formulations for Freeze-Drying

Kevin R. Ward and Paul Matejtschuk

Abstract
One of the most wide-reaching changes in freeze-drying over the past 20 years has been the commercial
availability of a range of discriminating analytical methods to identify critical temperatures in formulations
to be freeze dried. Here, we briefly describe the development of these techniques from the earlier bespoke
methods, setting their rise in a historical context. We then review techniques such as freeze-drying
microscopy, differential scanning calorimetry, and less widely applied alternative technologies. Practical
examples of the results obtained with these techniques are discussed and upcoming new trends and
troubleshooting problems are addressed.

Key words Freeze-drying microscopy, Differential scanning calorimetry, Dynamic mechanical

analysis, Thermal analysis, Impedance, TASC, Optical coherence tomography

1 The Basis for Formulation Analysis

1.1 Introduction Given their prominence in modern publications on freeze-drying, it

is perhaps surprising to realize that the use of modern thermal
analysis and techniques such as freeze-drying microscopy (FDM)
in formulation development was for the most part neglected until
the start of the twenty-first century. These technologies were devel-
oped by freeze-drying pioneers such as the late Prof Louis Rey [30]
and Alan MacKenzie [20] but were yet to be commercially
exploited. Although widely described as early as the 1960s such
techniques were very much the domain of the specialist laboratory
and many undertaking freeze-drying in academic or even industrial
laboratories relied upon a trial-and-error approach to cycle devel-
opment, sometimes supplemented by home-built simple analyzers.
For instance, at NIBSC in the late 1990s, there was a home-built
differential thermal analyzer built in conjunction with Brunel Uni-
versity, results from which went by the rather lengthy term of
“solid-liquid transition temperature” and this was calibrated with
isotonic saline, much as used with current thermal analyzers. To its
advantage, it required only 0.2 mL of sample; however, the results

Kevin R. Ward and Paul Matejtschuk (eds.), Lyophilization of Pharmaceuticals and Biologicals: New Technologies and Approaches,
Methods in Pharmacology and Toxicology, https://doi.org/10.1007/978-1-4939-8928-7_1,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

1
2 Kevin R. Ward and Paul Matejtschuk

were plotted on graph paper—a far cry from modern technology.

Quite common at the time was freeze-drying formulation based
solely upon trial-and-error experiments (see [22]) without any
formulation analytical characterization and these time-consuming
studies meant that formulation was often developed slowly with
“one factor at a time” (OFAT) approaches. This seems a long way
from the modern analytical laboratory where thermal and optical
methods vie for predominance and analytical characterization is a
prerequisite to anything being loaded into a freeze dryer!
The history of these developments and of others which still
remain the domain of the specialist laboratory have been previously
described by Rey [32, 33]. In this chapter, we will review the
common technologies used for formulation characterization to
optimize freeze-drying and also highlight some of the newer tech-
nologies that have recently come to the market place.

1.2 History of Using As stated previously, the development of these technologies has
Analytical Formulation been reviewed by some of those who initially developed them
[33]. Smith has also reviewed the history of one area of such
technology—that of impedance technology (see Chap. 11 in this
volume). The first such techniques were recommended by Rey
[30, 31] and Lachman et al. [18]. MacKenzie [20, 21] and also
Rey [31] described bespoke freeze-drying microscopes. The tech-
nology of freeze-drying itself was reviewed on several occasions by
the Developments in Biologicals series [4, 23].
The disadvantage of thermal analyzers is that they respond best
with salty ionic solutions, but these formulants are not the best for
optimizing freeze-drying. Although largely applied to aqueous
samples, analysis of mixed organic phases can also be analyzed [16].

1.3 Advantages of The advantages of performing thorough pre-lyophilization analysis

Pre-lyophilization of samples intended for drying are quite easily appreciated. The
Analysis amounts of material required are small compared to that needed for
a reproducible freeze-drying run, and the time taken even for slow
scan runs is orders of magnitude shorter than a freeze-drying run
(minutes rather than tens of hours). In addition, by combining
several techniques, a comprehensive analysis of the spectrum of
thermal and rheological properties can be accumulated within a
comparatively short time. This will then make a significant saving in
terms of the number of freeze-drying trials which will be needed
before arriving at a viable scalable lyophilization cycle. This must be
offset against the cost of the analytical equipment, which is not
insignificant, in the range of $25K–$100K per item. Some of these
techniques are highly specific, such as a freeze-drying microscope;
however, other techniques (e.g., DSC, DMA) can be applied in
other areas of pharmaceutical development and so the effective cost
may be offset in organizations with a broader formulation remit
than just a freeze-dried format.
 Characterization of Formulations for Freeze-Drying 3

2 Techniques Used to Characterize Formulations

2.1 Electrical The premise of using electrical properties to identify changes in

Impedance (Zsinφ) frozen solutions prior to (and even during) lyophilization was
Analysis discussed by Rey in 1960; however, the idea was never widely
commercialized, possibly because electrical resistance
(or resistivity) analysis seemed to display little sensitivity to solu-
tions containing much lower levels of ionic or polar species. This
was often the case with many biopharmaceutical and biological
products where buffers or other salts would typically be signifi-
cantly lower than in traditional injectable drug molecules
(or sometimes absent altogether) and even in the liquid state, the
electrical resistance would sometimes not be zero.
Rey demonstrated in 2004 that examining impedance (and in
particular, a function of impedance known as “Zsinφ”) rather than
simple resistance would typically give a much clearer signal of
mobility changes occurring in the frozen state. Indeed, for a num-
ber of different solutions, plotting the value of Zsinφ (in Ohms)
against temperature during cooling and warming often gave an
obvious indication of a softening event, even when such events
could not be easily detected by traditional thermal methods such
as DSC. After examining Zsinφ at a series of input frequencies
between 300 and 3000 Hz for a range of aqueous solutions, it
was concluded that a frequency of 1000 Hz gave the most accept-
able balance between amplitude of signal and clarity of event. An
industrial collaboration with Biopharma Process Systems enabled
Rey to commercialize the technology in the form of the Lyotherm2
instrument in 2005. We have employed this form of analysis very
widely in our own laboratories at BPS and NIBSC since that time,
particularly after much of the early data indicated that for more
complex formulations, there can be marked changes in mobility at
temperatures far lower than appear to translate to an obvious visible
change in the material when observed using freeze-drying micros-
copy (FDM) [39]. The thermogram containing the Zsinφ and
DTA profiles for a human plasma reference sample is shown in
Fig. 1.

2.2 Differential Differential Thermal Analysis (DTA) is a means of analyzing endo-

Thermal Analysis thermic and exothermic events in a sample by comparing its ther-
(DTA) mal behavior with that of a suitable reference material—in the case
of a liquid formulation, the reference material would typically be
the solvent (such as water). Being a relatively simplistic method, it
has largely been superseded by DSC (see later in this chapter) for
dry state analysis, due to the enhanced accuracy and sophistication
of the latter [17]. However, DTA does remain in use today in
lyophilization R&D, particularly for analysis of liquid samples pre--
lyophilization, where, for example, in modern instruments such as
4 Kevin R. Ward and Paul Matejtschuk

Fig. 1 Lyotherm Impedance and DTA profile for a human plasma reference sample. On the impedance (pink)
curve, the flattening at point Z1 indicates a stabilization within the structure upon warming, which ties in with
the minor thermal event at D1 in the DTA curve. The increase in downward gradient at point Z2 represents an
increase in molecular mobility within the frozen formulation, culminating in minimum impedance being
reached at
20  C (Z3), which coincides with the temperature of total collapse reported for plasma

the Lyotherm3 (where it can run simultaneously alongside Zsinφ

analysis, as shown in Fig. 1), the relatively large sample size (3 mL)
can help overcome the traditional issues with lack of signal strength
often observed for the analysis of dilute aqueous solutions where
the relatively high energy thermal events associated with the water
itself tend to dominate the profile.

2.3 Freeze-Drying
Microscopy (FDM)
Even the first designs of FDM apparatus focused on the use of thin
samples sandwiched between cover slips, in order to minimize any
sublimation cooling effects, since the temperature of the sample
itself was not measured directly and by minimizing such effects, the
sample temperature and the measured temperature could then
be assumed to be near-identical. Resulting data could be validated
against standard solutions—usually crystallizing solutions such as
sodium chloride, which would have a known eutectic melting
temperature and would visibly melt over a very narrow temperature
range when warmed sufficiently slowly. The same principle is still
employed today, but with various incremental improvements in the
accuracy of measurement, the precision of control and the optical
quality.
A typical FDM experiment involves cooling the liquid sample
until it appears to be completely frozen, then applying a vacuum
 Characterization of Formulations for Freeze-Drying 5

and observing the structure immediately behind the drying front

(sublimation interface). If the structure remains visibly intact, then
the temperature is raised until the point is reached where defects are
evident in the structure; if, on the other hand, the structure is poor
or there is no drying structure visible at all when sublimation
commences, then the temperature is lowered in order to determine
whether evidence of drying structure can be obtained. Examples of
lyophilization-related events that can be identified using FDM are
discussed in the sections below.

2.3.1 Collapse The term “collapse” is often used generically to describe any visible
loss of structure in a material undergoing freeze-drying; however,
strictly speaking, it refers to viscous flow in an amorphous—or
partially amorphous—material, as distinct from an eutectic melt
that would occur in a crystalline sample (or crystalline phase of a
“mixed system”). Ultimately, either form of event may be consid-
ered to produce a process defect in a product, although the mani-
festation of collapse and melting can be visibly different and may
have differing consequences on the product, process, and equip-
ment. An example of this is that a crystalline sample having under-
gone an eutectic melt (meaning there will be eutectic liquid
between the solvent crystals) may then see the liquid component
boil under the levels of vacuum employed during the sublimation
process, which may also lead to a loss of pressure control; on the
other hand, an amorphous sample undergoing collapse will become
more resistant to vapor flow (Rp increases) due to pores closing
down and surface area reducing, and therefore will not tend to lead
to loss of pressure control.
Since virtually all formulations of pharmaceuticals, biopharma-
ceuticals, vaccines, and medical/environmental diagnostics are at
least partially amorphous (with most being completely amorphous
apart from those containing small drug molecules and/or crystal-
line excipients such as mannitol), collapse is an event that is relevant
to most products [26]. Therefore, for most freeze-drying scientists,
the primary function of FDM is to determine the collapse tempera-
ture of a formulation. After gaining some experience of working
with lyo-formulations, it may be possible to predict what the col-
lapse temperature may be, although this can be somewhat challeng-
ing for formulations containing a mixture of crystallizing and
amorphous components, since the latter may inhibit (or even pre-
vent) the crystallization of the former, and this will also be depen-
dent on the ratios of the components, and often also on the
freezing conditions employed.
In our laboratories, we often carry out FDM analysis on a
sample multiple times, and as a minimum, for samples that display
low collapse temperatures when initially analyzed in the absence of
an annealing step, analysis will be repeated with the inclusion of an
annealing step, in order to determine whether this may have a
6 Kevin R. Ward and Paul Matejtschuk

positive impact on the thermodynamic stability of the material. This

is discussed further later in this chapter. Additionally, it may be
advisable to run thermal/impedance analysis prior to FDM in order
to enable a suitable temperature to be selected for the annealing
step in FDM.
Figure 2 shows images of sucrose solutions during FDM analy-
sis. If the sample is warmed slowly, or the temperature setpoint
increased in small steps (e.g., 0.1  C) after a period of holding in
each step, then the determination of the onset of the collapse event
should be possible to within a fraction of a degree. This is enabled
by the acquisition of images at short intervals (often taken as
frequently as every 5 s during certain steps of the analysis), which
allows for retrospective analysis of images where the aim is to find
two consecutive images in the resulting gallery, one of which shows
the sample to have perfect appearance and the subsequent one
showing the first signs of imperfection. The ability to pinpoint the
precise onset of collapse may be enhanced through the use of an
analyzer collar and tint plate assembly; visualizing a sample with
polarized light and a first order red filter enables in-depth analysis of
crystal structures in the frozen material, while the false color also
allows for easier identification of critical events.

2.3.2 Microcollapse Microcollapse has been an identifiable phenomenon in its own right
for a number of years, as discussed by Wang [38] and Liu et al. [19]
and is one that we have frequently observed in our laboratories in
formulations containing a mixture of crystalline and amorphous
components (which may be excipients and/or the active ingredi-
ent). For example, a solution containing 2% mannitol and 1%
glucose where the mannitol crystallizes during freeze-drying
(either as a controlled event during a deliberate annealing step, or
perhaps in a less controlled manner during sublimation) would be
expected to comprise separate phases in the frozen (and drying)
structure where mannitol exhibits a Teu of
1.4  C but glucose
undergoes viscous flow around
41  C, thus microcollapsing onto
the scaffold of crystalline mannitol (see Fig. 3). In this case, a
conservative estimate of the critical temperature might be consid-
ered to be
41  C, and indeed, some defects in the freeze-drying
structure are clear at this temperature when viewed by FDM
(Fig. 4), although we have observed that in such a mixture, total
loss of structure at the macroscopic level often does not occur until
above
20  C. This is still significantly lower than the eutectic
temperature of crystalline mannitol alone; whether this is attribut-
able to significant levels of viscous flow in the amorphous glucose
phase at this temperature that was able to counter the mechanical
strength afforded by crystalline mannitol, or whether indeed some
of the mannitol persisted in the amorphous phase due to the
presence of amorphous glucose and/or the thermal history of the
Fig. 2 Examples of FDM images taken at various key points during the analysis of disaccharide samples, in
each case frozen to
40.0  C initially, then once vacuum applied, gradually warmed until loss of structure
observed: (A) sample immediately before onset of collapse occurs, (B) the same sample 6 min later, having
been warmed to the point of complete collapse, and (C) demonstrating the visual effect of using a polarizer
and first order red filter (right) compared with using no filters or polarization (left) throughout FDM analysis
8 Kevin R. Ward and Paul Matejtschuk

Fig. 3 Cartoon representation of microcollapse, with crystalline phase

(represented by black lines) remaining structurally intact but amorphous phase
(shown in red) collapsing when the sublimation interface temperature exceeds
the Tc of this phase during primary drying. Micro-melting, as defined by us
previously [39], would be analogous to microcollapse but with the amorphous
phase remaining structurally intact and crystalline phase undergoing eutectic
melt when the Teu of this phase is exceeded

Fig. 4 Typical FDM image showing microcollapse in a real sample, which is

manifested in a “multi-layer” semi-opaque appearance with the crystalline
phase retaining its structure but the amorphous phase collapsing onto
it. Evidence of this observation as being microcollapse (as opposed to the
onset of a classic collapse event) may be confirmed if there is no further loss
of structure when the temperature is increased very slightly
 Characterization of Formulations for Freeze-Drying 9

sample, was not clear. This example illustrates the point that com-
bining crystalline and amorphous components does not always give
a single critical temperature that can be predicted from the behavior
of the individual components, and that exceeding a particular ratio
could lead to step changes in critical temperature with respect to
the macroscopic structure due to phase/state changes in one or
more of the components, which may also be a function of thermal
history. Similar observations were made by Adams and Irons [1] for
mixtures of sodium chloride and lactose.
Conceivably, in mixtures where the collapse temperature of the
amorphous phase is higher than the eutectic temperature of the
separate but coexistent crystalline phase could lead to an analogous
phenomenon that we have notionally termed “micromelting” [39],
where a crystalline formulation component (or combination of
such components) with a low eutectic temperature melts onto the
“backbone” of a rigid amorphous phase within the material. An
example of this that has been observed in our laboratories is that of
a biological product containing a small amount of calcium chloride
as part of a buffer system; in this case, while macroscopic collapse
was not observed until temperatures above
30  C (by FDM,
unpublished data), the use of other methods suggested significant
mobility changes around
53  C, the eutectic temperature of
calcium chloride. When the formulation was freeze-dried above

53  C, no shrinkage was observed at the macroscopic level,
although there was evidence of heterogeneity in the form of small
visible “spots” on the base of the cake.

2.3.3 Eutectic Melting For samples that are largely or wholly crystalline, the eutectic
melting temperature will be the critical temperature above which
defects will occur in lyophilization. This phenomenon can be easily
identified using a number of analytical methods, including FDM.
Figure 5 shows a sample of sodium chloride solution drying below
and above its eutectic temperature, illustrating the fact that the
eutectic temperature can be identified very clearly using FDM,
due to the marked visible change in sample appearance that occurs
at Teu. This includes the previously frozen part of the sample
appearing visibly as a “partial liquid” and also the contraction of
this part of the sample which then recedes away from the previously
dried material.

2.3.4 The Effect of The technique of annealing (also called heat-annealing or temper-
Annealing ing) as part of the thermal treatment segment of a lyophilization
cycle has been well established as a means of influencing the ice
structure and/or encouraging crystallization—or polymorphic
changes—in solutes. It is outside the remit of this chapter to discuss
the physicochemical theory and basic principles of the processes
occurring in annealing, which have been covered previously in
10 Kevin R. Ward and Paul Matejtschuk

Fig. 5 FDM images showing a frozen solution of sodium chloride drying (A) below
and (B) slightly above its eutectic temperature

other texts, but to highlight the information regarding the practical

effects of the annealing process that can be observed for real for-
mulations using FDM.
Figure 6 shows the visual effect of annealing a sample of frozen
formulation (initially frozen to
40  C) at
10  C for 10 min.
While in this example it is not possible to quantify the extent and
speed of ice crystal growth, FDM experiments employing different
conditions (temperatures and holding times) can be carried out and
the relative differences established, in order to direct the user
toward the selection of the most suitable or effective annealing
conditions in a real lyophilization scenario.
Figure 7 illustrates the fact that following the initial nucleation
and ice crystal growth phase, it is not necessarily possible to
 Characterization of Formulations for Freeze-Drying 11

Fig. 6 FDM images of a frozen sample immediately prior to (A) and after 10 min
of (B) annealing at
10  C, depicting the visible change in appearance, related
to increasing ice crystal size and networking. Light level and focus remained
unchanged during this time

determine whether the solute has crystallized, but that upon hold-
ing the sample for an extended period, solute crystallization may be
confirmed by the observation of a second freezing event—in this
case, the appearance of a “crystallization front” that sweeps
through the amorphous solute. A similar effect may be observed
more quickly by the use of annealing, with the general rule of
thumb that this is accelerated considerably when the amorphous
material is held above its characteristic glass transition temperature
(Tg0 ), which may be identified using thermal methods as discussed
in other sections of this chapter.
12 Kevin R. Ward and Paul Matejtschuk

Fig. 7 Extended holding of a sample at
40  C eventually leads to the

appearance of a crystallization front (A) which then moves slowly through the
frozen amorphous material (B)

2.3.5 Formulations There are many drug products on the market that are freeze-dried
Containing Organic from organic solvents or co-solvent mixtures (usually mixtures of
Solvents water + an organic solvent). The lyophilization of such formula-
tions brings its own challenges, not least in achieving complete
freezing and avoiding phase separation, the control of the sublima-
tion process and establishing whether the co-solvents behave as
azeotropic or zeotropic mixtures, residual solvent levels allowable
in the final product, and the safety and environmental issues of
handling, condensing, and reclaiming the organic solvents them-
selves. A comprehensive review of such challenges was provided by
Teagarden et al. [36] and it is not the intention of this chapter to
reiterate these points here, but again to highlight how the use of
 Characterization of Formulations for Freeze-Drying 13

Fig. 8 FDM image of a co-solvent-based formulation following freezing and the

application of vacuum, showing evidence of egress of the (more volatile) organic
solvent from certain areas (examples highlighted by red circles, with direction of
solvent vapor movement indicated by dashed arrows)

FDM can provide information as to the practical aspects of freeze-

drying a formulation containing multiple solvents. Figure 8 shows a
sample undergoing sublimation during FDM analysis where the
organic solvent is visibly being removed via its own network of
channels, while the frozen aqueous component dries from the
edge of the sample in the same way as if it were a purely aqueous-
based sample. This observation suggests that phase separation of
the two solvents has taken place, which may indicate that there may
be an opportunity to remove the two solvents sequentially (espe-
cially if the vapor pressures are sufficiently different), although care
should be taken if the solutes have partitioned into the different
solvent phases, as this may lead to microcollapse or micromelting
within the structure.

2.3.6 Formulations It has been noted that certain formulations are particularly suscep-
Susceptible to Skin/Crust tible to forming a surface skin (crust) on freezing, which will
Formation subsequently impede the sublimation process or even prevent dry-
ing altogether. Since none of the traditional thermal methods is
able to pick up on the potential for this phenomenon to occur, it is
rarely noticed until post-lyophilization inspection of the product
appearance. However, FDM can sometimes highlight such behav-
ior, as was the case for the sample shown in Fig. 9. Samples
exhibiting a particular propensity to form a skin or crust tend to
be dilute solutions, where ostensibly the viscosity of the liquid
formulation at the moment of ice nucleation is very low and conse-
quently, the physical action of ice crystal growth results in the solute
14 Kevin R. Ward and Paul Matejtschuk

Fig. 9 Example of skin (crust) formation during freezing, resulting in a dense

concentrated wall of solute at the edge of the sample and reluctance to dry until
the skin ruptures (A), whereupon the action of sublimation results in further
breakup of the skin, allowing drying to progress more quickly but with an uneven
sublimation front (B)

being forced to the edge (or surface) of the liquid as it solidifies.

This phenomenon can also be commonly observed when freezing
solutions containing particular proportions of certain salts and
sugars, such as a solution of 1% lactose + 1% sodium chloride.
Skin or crust formation can sometimes be avoided by altering the
total solute concentration, varying the proportions of components
or using a controlled nucleation method to instigate “top down”
freezing (as discussed in Chaps. 3 and 4 of this volume). If the
 Characterization of Formulations for Freeze-Drying 15

phenomenon cannot be avoided altogether, then it may be possible

to increase the porosity in the skin or crust by including an anneal-
ing step, if indeed annealing is compatible with the formulation in
question. Alternatively, heterogeneity may be reduced by using
rapid initial cooling during the freezing process, which although
is likely to give smaller ice crystals and thus increased product
resistance (Rp) to vapor flow, the value of Rp may still be lower in
this case than the equivalent value in the presence of the skin or
crust.

2.3.7 Formulations One of the more recent challenges we have faced is in the lyophili-
Containing Glycerol zation of formulations that contain glycerol, such as with some
PCR (polymerase chain reaction) reagents. The PCR-based
method of detection in In Vitro Diagnostics (IVDs) is becoming
the method of choice in many applications compared with enzyme-
or antibody-based detection systems. Historically, the standard
storage conditions for PCR reagents have been at low temperatures
(often
80  C) but typically after the addition of glycerol to act as
somewhat of an “antifreeze,” thus arguably reducing the risks
associated with the process of freeze-concentration, but at the
same time, minimizing degradation rates by the use of low tem-
peratures, as defined by Arrhenius kinetics. Naturally, any antifreeze
effect gives such formulations an obvious lack of compatibility with
the lyophilization process! We have observed this to be possible in
some cases in our laboratories when the glycerol content is suffi-
ciently low (typically less than 0.2% but also depends on the pro-
portion of glycerol to other components). However, what often
happens is that even when ice is able to form, the glycerol remains
unfrozen and becomes forced toward the edges of the sample, as
shown in the FDM images in Fig. 10. In the classical lyophilization
situation where ice tends to form at the base of a solution
(or suspension), this would translate to the glycerol being pushed
to the surface of the frozen mass, where it will persist as an “oily”
liquid that obstructs the sublimation process. Controlled nucle-
ation systems may offer an opportunity to circumvent this issue,
but the evidence for this remains yet to be seen.

2.3.8 Reducing While operators can gain experience and confidence quite rapidly
Subjectivity in FDM with modern FDM systems, there is still the desire to have a means
Experiments: The Use of independent verification of collapse events in order to minimize
of TASC subjectivity as far as is possible in such a patently optical method; it
was this need for reduced levels of subjectivity that has led to the
development of TASC—Thermal Analysis by Structural
Characterization.
TASC works by analyzing a sequence of images and tracking
the changes from one to another. First, the operator selects the
range of the analytical run and clicks on the TASC option, as shown
in Fig. 11.
16 Kevin R. Ward and Paul Matejtschuk

Fig. 10 Evidence of an excluded layer of glycerol on the edges of frozen samples,

preventing drying from occurring in both cases. In (A), the liquid is indicated by
the arrow, while in (B) the liquid layer is more obviously visible, with further
evidence of ice or solute crystal growth extending into the glycerol layer

Second, a “region of interest” is selected (shown in red in

Fig. 12). This area will be the part that is tracked and covers the
sublimation front and the material to be dried ahead of it. Third,
the “region to be scanned” is selected (shown in blue in Fig. 12).
Scan size should be kept relatively small to minimize processing
time, but TASC analysis typically takes less than 5 min.
The output of the analysis is a graphical plot, as shown in
Fig. 13, which illustrates how the images at each TASC transition
can be viewed by clicking on the associated image (1) to see how
they are relevant. The two points of interest, in this case, are where
the gradient of the line changes (2) and the maximum (3). Images
during this analysis were captured every 5 s at a heating rate of
Another random document with
no related content on Scribd:
Wenn auch nach J e n t i n k s Klarstellung der Unterschiede von C.
marginatus und brachyotis (1891) eine Revision der Bestimmungen von
marginatus in den Museen angezeigt ist, so scheint es doch, nach den
Catalogen des Britischen und des Leidener Museums (1878, 83 und
1888, 153), nicht zweifelhaft, dass sich überall, wo brachyotis
vorkommt, auch marginatus findet, und so sind vielleicht die Acten über
das Verhältniss der beiden Arten zu einander noch nicht zu schliessen.

[Inhalt]

11. Uronycteris 3 cephalotes (Pall.)

fem.,
a, b.
in Spiritus, Kema, Minahassa, Nord Celébes, 93 (69,
65 mm).
fem.,c.
in Spiritus, Makassar, Süd Celébes, IX 95 (67 mm).

J e n t i n k (NLM. 1883 V, 173) hat von einem adulten Männchen von

Amurang, Minahassa, bemerkt, dass es grösser sei als gewöhnlich,
nämlich (Vorderarm) 67 gegen 61 mm (2.4 inch.), was D o b s o n (Cat.
1878, 90) als constantes Maass adulter Exemplare aus dem
Ostindischen Archipel angiebt. Dann hat H i c k s o n (Nat. N. Cel. 1893,
84), dem wohl J e n t i n k s Bemerkung unbekannt geblieben ist, gesagt,
dass die Celébes-Exemplare längere Vorderarme hätten als die von
irgend einer anderen Localität, er giebt (p. 359) an: 63–76 für Manado
und 56–65 für Ternate, Ambon, Timorlaut, Cap York und die Admiralitäts
Inseln (?). Die Dresdner Exemplare von der Minahassa (66, 67),
Gorontalo (68) und Makassar (67) sind ebenfalls grösser, sie (4)
variiren, zusammen mit den 3 S a r a s i n schen, von 65–69; eins von
Siao misst sogar 75 (3 von der Nordosthalbinsel von Celébes, den
Inseln Manado tua und Talaut gestatten dieses Maass nicht zu
nehmen), eines von Ternate nur 56.
H i c k s o n knüpft an die grösseren Dimensionen der Exemplare von
Celébes die Vermuthung, „that the struggle for existence among bats is
so keen in Celebes, that only the extremely long-winged forms … have
been able to compete in the conditions of life“. Für Cephalotes peroni
(s. unten p. 9) aber zieht er für die angeblich geringeren Dimensionen
der Celébes-Exemplare dieselbe Schlussfolgerung.

Uronycteris cephalotes soll nach verschiedenen Quellen von Celébes

bis Morotai, Halmahéra, Gebeh, Ambon, Timorlaut, NW Neu Guinea
und Cap York zu Hause sein, während auf Misol, SO Neu Guinea,
Fergusson, Duke of York, Neu Irland und den Salomo Inseln U. major
(Dobs.) vorkäme, welche Art nach D o b s o n (Cat. 1878, 89) grösser
und heller ist als cephalotes, aber kürzere Ohren und längere
Nasenröhren hat, bei abweichendem Schädel und Zahnbau (PZS.
1877, 117 Abb.). Nun giebt D o b s o n den Vorderarm von major auf 78
mm (3.1 inch.) an, was dem Celébes-Maasse bis 76 bei cephalotes
(H i c k s o n ) und dem [9]von Siao mit 75 ganz nahe kommt. Wenn er
von major sagt: „upper canine with a prominent cusp“, von cephalotes
„with a blunt ill-defined external projection“ (s. auch Fig. 2 a und 3 a, l.
c.), so muss ich dazu bemerken, dass dies nicht durchgreifend ist, denn
ein Männchen von Amurang, Nord Celébes, im Dresdner Museum (Nr.
683) zeigt den major-Charakter, bei einer Vorderarmlänge von 66 mm.
Was die Färbung angeht, so sagt D o b s o n von der Unterseite von
cephalotes (Cat. 89): „dull yellowish white“ und von der von major (p.
90): „dull yellowish buff throughout“. Exemplare von cephalotes von
Tonkean (NO Celébes), Siao und Talaut im Dresdner Museum aber sind
keinenfalls dull yellowish white, soweit man derartige
Farbenbezeichnungen beurtheilen kann. Es giebt hellere und dunklere
Exemplare aus der Minahassa, die eben erwähnten von Tonkean etc.
aber sind eher „raw umber“ oder „tawny-olive“ (Ridgway Pl. III, 14 und
17), also auch nicht „dull yellowish buff“; auf der anderen Seite stimmt
ein mir vorliegendes Exemplar von major von Fergusson Is. in der
Farbe der Unterseite genau mit einem von cephalotes von Gorontalo in
Celébes, wenigstens wie letztere Art bis jetzt angesehen wurde. U.
cephalotes variirt, wie viele Arten in der Färbung je nach dem Alter,
worauf schon P e t e r s (Mb. Ak. Berlin 1867, 868) aufmerksam
gemacht hat.

Wenn ich nun auch nicht dahin neige, den Werth von major als
Subspecies anzuzweifeln, so ist doch, auch angesichts der bis jetzt
bekannten geographischen Verbreitung der beiden Formen, die
Sachlage unklar. Da die grosse U. major auf Misol vorkommen soll (Cat.
MPB. XII, 186) und auf Celébes nebst Siao eine Form, die etwas
grösser ist als die typische kleine cephalotes der dazwischen liegenden
Fundorte (Ternate, Halmahéra, Ambon), so müssten auch diese zwei
Formen von cephalotes einander und major subspecifisch coordinirt
werden. Allein das Material der Museen ist noch zu ungenügend, um
hier festen Fuss fassen zu können; dazu wären nicht nur viel mehr
Exemplare von den bereits bekannten Fundorten nöthig, sondern auch
solche von den zwischen Celébes und SO Neu Guinea liegenden
Gegenden, von denen noch Nichts bekannt ist. Erst dann wird man
urtheilen können, welcher Werth der U. major zukommt, und ob auch
das Celébes-Areal eine Subspecies beherbergt.

[Inhalt]

12. Cephalotes peroni Geoffr.

fem.,
a, b.
Bälge, Tomohon, Minahassa, Nord Celébes, 10. X 94
(117, 113 mm).
fem.,c.
in Spiritus, Tomohon (100 mm).
mas,d.in Spiritus, Kottabangon, Bolang Mongondo, Nord
Celébes, 2. XII 93 (116 mm).
fem.,e.in Spiritus, Buol, Nord Celébes, VIII 94 (95 mm).
Die Art findet sich von Celébes bis zu den Salomo Inseln. Auf Celébes
selbst ist sie vom Norden und Süden bekannt. Vom Norden von
Amurang 4 in der Minahassa und von Gorontalo (Mus. Leid.),
desgleichen und von Manado (Mus. Dresd.), wozu noch die obigen
S a r a s i n schen Fundorte kommen; vom Süden von Makassar (Mus.
Dresd.); auch J e n t i n k s Exemplar p (Cat. XII, 156) ist aus Süd
Celébes, da Te i j s m a n n 1877 in Süd Celébes (und Saleyer)
sammelte (s. NTNI. 1879, 54). Das Dresdner Museum hat die Art ferner
von Sangi und Talaut, von wo sie noch nicht registrirt war.

H i c k s o n (Nat. N. Cel. 1893, 85 und 359) sagt, dass die Exemplare

von Manado im Durchschnitte kürzere Vorderarme hätten als die aus
andern Theilen des Archipels, und zwar 104 mm von Manado gegen
103–151 von anderswo, allein er giebt weder an, wie viele Exemplare
von Manado er gemessen hat, noch ob sie adult waren; letzteres
bezweifle ich, da ein S a r a s i n sches (c) 117 und ein Dresdner von
Amurang 115 misst; seine Behauptung ist so ungenügend fundirt, dass
ihr keine Beweiskraft zukommt, und damit fällt auch die daran geknüpfte
Schlussfolgerung (vgl. oben bei Uronycteris cephalotes p. 8). Weit
entfernt eine an die Localität gebundene Differenz in den Maassen des
Vorderarms a priori in Abrede stellen zu wollen, so gehört doch, meine
ich, zu ihrer Constatirung eine ganz andere Grundlage.

D o b s o n beschrieb 1878 (PZS. 875) eine zweite Art der Gattung

Cephalotes, C. minor, von Amberbaki, Nordwest Neu Guinea und sagt,
sie sei halb so gross wie C. peroni, sonst gleich, nur mit weniger spitzen
Ohren und viel kleineren Füssen, auch setze die Flügelmembran an der
äusseren Zeh und tiefer an, und die Zähne seien „slightly different“. Das
Dresdner Museum besitzt ein s e h r grosses Exemplar [10](Balg) von
der Insel Mansinam bei Doré, Nordwest Neu Guinea, das den
angeführten Charakter der Flügelmembran exquisit aufweist, während
die anderen angegebenen Unterscheidungsmerkmale hier nicht
zutreffen; die ganze Länge (Kopf und Körper) ist c 225 mm, der
Vorderarm 148. Ferner ein kleines Exemplar von der Astrolabebai,
Südost Neu Guinea (in Spiritus), das ebenfalls den abweichenden
Flügelmembranansatz zeigt; ganze Länge c 100 mm, Vorderarm 70.
Dagegen ist ein Exemplar von der Insel Mysore in der Geelvinkbai in
dieser Beziehung typisch wie C. peroni und ebenso verhalten sich die
Exemplare von Ternate und Ambon. Dass die bis jetzt bekannten 3 Neu
Guinea Exemplare nur zufällig jenen unterscheidenden Charakter
aufweisen sollten, ist auszuschliessen; welche Bedeutung ihm aber, bei
den nicht stichhaltigen anderen von D o b s o n aufgeführten
Unterschieden, beizumessen ist, wird erst die Zukunft lehren.

[Inhalt]

13. Carponycteris australis (Ptrs.)

mas,a.in Spiritus, Kema, Minahassa, Nord Celébes, 21. VIII

93 (39 mm).
fem.,
b–e.dgl. (39 mm).

P e t e r s benannte (Mb. Ak. Berl. 1868, 13 Anm.) eine kleinere

Carponycteris-Art von Rockhampton, Ost Australien, gegenüber der
grösseren minima (Geoffr.), als var. australis und führte sie später (l. c.
p. 871) als Art auf mit dem Bemerken, dass es noch fraglich sei, ob
man es mit einer Art oder einer Localrasse zu thun habe (er sagt da,
irrthümlicherweise, von West Australien). D o b s o n (Cat. 1878, 96)
citirt zwar P e t e r s unter Macroglossus minimus, ignorirt aber australis
und giebt die Verbreitung von minima als von Darjeeling bis zu den
Philippinen, Nord und West Australien und Neu Irland; T h o m a s
dagegen (PZS. 1888, 476) nennt australis von den Salomo Inseln und
sagt, die Art unterscheide sich von minima auch durch das tief
gefurchte Rhinarium (die sonstigen Unterschiede im Gesichte, die
T h o m a s anführt, — Gesicht und Oberlippe kürzer — kann ich an
dem Dresdner Materiale nicht auffinden), und sie gehe bis zu den
Philippinen (1898 TZS. XIV, 385 führt er sie auch von Negros auf, und
M a t s c h i e Sb. ntw. Fr. Berl. 1898, 39 von Tablan), Mysol und Duke of
York; die Vorderarmlänge von minima (10 Ex.) sei 38–43 mm, die von 5
javanischen Exemplaren 45–48 (später hat T h o m a s noch eine Art:
crassa von den Fergusson Inseln beschrieben, die aber nicht kleiner ist
als minima, NZ. 1895, II, 163). B l a n f o r d , der T h o m a s auf einen
wesentlich unterscheidenden Charakter von australis aufmerksam
gemacht hat (PZS. 1888, 476 Anm.), sagt in seiner Fauna von Britisch
Indien (Mam. 1888, 265) noch, dass es nur éine Art Carponycteris
gäbe.

Über die Zugehörigkeit der Nord Celébes-Exemplare der Herren

S a r a s i n zu australis waltet für mich kein Zweifel ob. J e n t i n k führte
zwar (NLM. 1883 V, 174, 1888 XI, 29 und Cat. MPB. 1888 XII, 159)
minima von Nord Celébes auf, allein dies war, ehe T h o m a s die
Unterschiede von australis klar gelegt hatte. Das Maass der
Vorderarme mit 39 mm und das gefurchte Rhinarium weisen den
Celébes-Exemplaren ihre Stelle an. Das Dresdner Museum besitzt
australis ferner von Sangi (39 mm), Nordwest Neu Guinea (42), Aru
(38.5) und Murray Insel (38) — es sind hier in Parenthese immer nur die
Maximalmaasse angegeben —, die Vorderarme variiren also von 38–42
(T h o m a s 38–43), während die Exemplare von Java und Sumátra
(minima) von 44.5–46.5 (T h o m a s 45–48) variiren.

Bei dem noch so mangelhaften Materiale der Sammlungen lässt sich

heute nicht festlegen, wo die geographische Grenze zwischen C.
minima und der Subspecies australis zu ziehen sei, speciell Bórneo
steht noch aus, aber es scheint, dass die Festlandsform minima sich bis
Java erstreckt, und dass australis von den Philippinen und Celébes bis
zu den Salomo Inseln und Ost Australien verbreitet ist. [11]

1 Bei den Fledermäusen sind (in Parenthese) die Vorderarmmaasse angegeben, auf
die stets, als charakteristisch, Werth gelegt wurde; neuerdings machte J e n t i n k
(Webers Zool. Erg. I, 125 1891) noch besonders darauf aufmerksam, dass es besser
sei, dies Maass zur Beurtheilung des Alters des Individuums anzuführen, als die
Bezeichnungen adult, semiadult, juv. etc. Man darf dabei aber nicht übersehen, dass
ein exactes Messen des Vorderarms nur am Skelette möglich ist, wo man den Radius,
die Ulna und den Sesamknochen der Tricepssehne gesondert vor sich hat. Bei Bälgen
ist es schwer und oft gar nicht möglich, das proximale Ende der verkümmerten Ulna
zu tasten und es von dem Sesamknochen zu trennen. Auch bei Spiritusexemplaren ist
es nicht leicht. Das empfehlenswertheste Maass wäre das des Radius, der bei den
Fledermäusen so vorzüglich entwickelt ist, aber auch dies wäre an Bälgen und
Spiritusexemplaren oft schwer oder unmöglich exact zu nehmen, da man füglich
weder sein proximales noch sein distales Ende bei jedem Exemplare freilegen kann.
Es ist daher unter der „Länge des Vorderarms“ stets nur ein ungefähres Maass zu
verstehen, was aber auch für den vorliegenden Zweck genügt. ↑
2 Xantharpyia J. E. G r a y List spec. Mam. Br. M. 1843, 37: Cynonycteris P e t e r s
Reise Mossamb. I Säugeth. 1852, 25. Schon B l a n f o r d (Fauna Br. Ind. Mam.
1888, 261) und T h o m a s (PZS. 1894, 449 etc.) haben sich für Xantharpyia
entschieden. ↑
3 T h o m a s brauchte 1895 (NZ. II, 163) Uronycteris, statt des bis dahin üblichen
Gattungsnamens Harpyia und sagte anmerkungsweise: „Lydekker; replacing
Harpyia …, preoccupied“, allein, so viel ich sehe, that L y d e k k e r dies nicht. Er hat
(F l o w e r & L.: Intr. Mam. 1891, 654) Carponycteris für Macroglossus eingeführt, aber
gebraucht (p. 653) Harpyia, und T h o m a s selbst kehrte 1896 (NZ. III, 526) zu
Harpyia zurück. Uronycteris rührt von G r a y her (PZS. 1862, 262). Harpyia Ill. (Chir.)
stammt aus dem Jahr 1811, Harpyia Ochsh. (Lep.) aus dem J. 1810, dieser Name
muss daher für die Fledermausgattung Uronycteris Platz machen. ↑
4 Im Cat. MPB. XII, 156 (1888) ist zwar Ex. o als von Menado aufgeführt, allein es ist
nach J e n t i n k NLM. V, 170 und 174 (1883) von Amurang. ↑
[Inhalt]
Microchiroptera
 Rhinolophidae

[Inhalt]

14. Rhinolophus minor Horsf.

mares,
a–c. in Spiritus, Kema, Minahassa, Nord Celébes, X
93 (41.5 — 41.5 — 40 mm).
fem.,d.in Spiritus, Kema, X 93 (42 mm).

Diese Art ist von Celébes noch nicht registrirt worden, Dr. R i e d e l
aber hatte sie schon im Jahr 1875 von Gorontalo nach Dresden
gesandt, und neuerdings kam sie auch von Talaut hierher (ein
Exemplar aus der Höhle von S. Mateo bei Manila ist vielleicht ein
wenig abweichend in der Form des Nasenbesatzes; minor ist sonst
von den Philippinen noch nicht aufgeführt). Dagegen ist die sehr
nahe stehende, grössere Rh. affinis Horsf. von J e n t i n k bereits
von Tondano, Nord Celébes, genannt worden (NLM. XI, 30 1888 und
Cat. MPB. XII, 162 1888), allerdings nur ein junges Weibchen. Nach
D o b s o n (Cat. 1878, 112 und 115) gehen beide Arten von
Vorderindien bis Bórneo. Bei diesem Parallelismus könnten beide
auch auf Celébes vorkommen, allein da J e n t i n k nur ein j u n g e s
Weibchen vorlag, so ist weiteres Material abzuwarten. P e t e r s gab
1872 (MB. Ak. Berl. 306) nur an, dass minor „ganz ähnlich affinis sei,
aber kleiner“ (den Fundort Timor bei minor glaubte er mit ?
bezeichnen zu müssen, J e n t i n k Cat. MPB. XII, 162 1888 aber
führte ihn wieder von daher auf). Es ist auch schwer, abgesehen von
der Grösse, durchgreifende Unterschiede aufzufinden, da minor
nach D o b s o n (Cat. 1878, 115) in Bezug auf die Sella, die
Interfemoralmembran und 2 pm inf. variirt. Die Grössenunterschiede
sind nach D o b s o n relativ ansehnlich, allein seine Maasse treffen
nach den Dresdner Exemplaren nicht überall zu. Das Verhältniss
dieser zwei Formen zu einander erfordert vielleicht eine gründliche
Untersuchung an reichem Materiale, wie es aber die Museen noch
nicht von überall her besitzen.

Die Färbung von minor ist nach D o b s o n (l. c. 114) hellbraun oben,
graubraun unten. Das erwähnte Exemplar von Gorontalo ist aber
„tawny“ (Ridgw. V 1) oben und „russet“ (III 16) unten, beide Nüancen
sogar noch lebhafter, allein da affinis in der Farbe variabel ist
(„greyish brown, reddish brown, golden orange brown“ D o b s o n l.
c. 112), so wird aller Wahrscheinlichkeit nach auch minor in der
Farbe variiren.

Sonst ist von Celébes noch Rh. megaphyllus Gr. aufgeführt worden,
und zwar von Manado (D o b s o n l. c. 112) und Amurang,
Minahassa, letzteres in 3 Exemplaren (J e n t i n k NLM. V, 174 1883;
Cat. MPB. XII, 161 1888 1 Ex.), von wo auch das Dresdner Museum
2 Exemplare hat, die von P e t e r s als Rh. euryotis Temm. bestimmt
worden sind; ich halte sie aber, so weit Sicherheit bei ausgestopften
Exemplaren möglich ist, eher für megaphyllus. Diese Art ist affinis
(und daher auch minor) nahe verwandt, D o b s o n (Cat. 1878, 111)
sieht sie als australische Repräsentantin von affinis an, und führt
eine var. α von Batjan und eine var. β von Nord Celébes und Goram
auf. Eine genauere Kenntniss der Form von Celébes und den
Molukken liegt noch nicht vor. Rh. euryotis steht den genannten
Arten ebenfalls sehr nahe und ist bis jetzt von Ambon, Ceram, Aru
und Kei bekannt. Eine Revision der ganzen Gruppe ist erwünscht,
allein die Materialien der Museen genügen auch dazu schwerlich.

[Inhalt]
15. Hipposiderus diadema (Geoffr.)

fem.,
a, b.
in Spiritus, Buol, Nord Celébes, VIII 94 (82, 80 mm).
fem.,c.
in Spiritus, Kalaena Thal, Luhu, Central Celébes, c.
200 m hoch, 4. II 95 (86 mm).

J e n t i n k wies zuerst diese von Vorderindien bis zu den Philippinen

und den Salomo Inseln, also sehr weit verbreitete Art von [Süd]
Celébes und Sula nach (Cat. MPB. XII, 166 1888) und dann von
Central Celébes (Webers Zool. Erg. I, 127 1890). Ich erhielt sie 1871
in Gorontalo (Mus. Berlin), und das Dresdner Museum besitzt sie
seit 1877 von Amurang in der Minahassa, sie kommt also, wie zu
erwarten, über ganz Celébes vor. Auch von der Insel Kalao im
Süden ist sie im Dresdner Museum, sowie von Talaut im Norden. [12]

D o b s o n (Cat. 1878, 137) giebt das Vorderarm-Maass auf 86 mm

(3.4 inch.) an, was mit obigen Maassen der S a r a s i n schen
Exemplare mehr oder weniger stimmt, J e n t i n k hatte von Luhu
eins von 92 mm (Webers Zool. Erg. I, 127 1890); die Dresdner von
Celébes messen selbst bis 93, die von Java bis 87, von Nordwest
Bórneo bis 86, von Südost Mindanao bis 83, von Süd Neu Guinea
bis 77 mm etc. Nur an der Hand eines grossen Materiales wird man
überhaupt der Frage näher treten können, ob diese Art von
Vorderindien bis zu den Salomo Inseln gar nicht variirt, was an und
für sich wenig wahrscheinlich ist.

Es kommt noch eine zweite, kleinere, Art von Hipposiderus auf

Celébes vor, H. bicolor (Temm.). Dresden besitzt sie seit 1877 von
Amurang im Norden, und J e n t i n k hat sie 1883 (NLM. V, 174)
ebendaher aufgeführt, und zwar als D o b s o n s var. α (fulvus Gr.);
das Dresdner Exemplar aber hat nicht die schöne goldgelbe
Färbung von fulvus, sondern ist oben ungefähr „Prout’s brown“
(Ridgw. III, 11), unten weisslich „wood brown“ (III, 19), also typisch,
die Art variirt demnach in der Farbe wie Rhinolophus affinis und
andere. H i c k s o n (Nat. N. Cel. 1893, 85) sagt von den Exemplaren
der kleinen Insel Talisse im Norden von Celébes, dass sie röther
seien, als irgendwelche im Britischen und Leidener Museum, sie
werden es aber, glaube ich, auf Talisse nicht zu allen Zeiten und
nicht alle sein. 1890 führte J e n t i n k die Art auch von Süd Celébes
auf (Webers Zool. Erg. I, 127 1890) und meinte, dass die
Vorderarmlänge von 41 mm viel grösser sei, als D o b s o n sie
angegeben, allein dieser hat (Cat. 150) 39.37 mm (1.55 inch.), also
eine unbedeutende Differenz; das Dresdner Exemplar von Amurang
misst 39, zwei von Nord Luzon ergeben 38–39 mm.
[Inhalt]
 Nycteridae

[Inhalt]

16. Megaderma spasma (L.)

fem.,a.in Spiritus, Tomohon, Minahassa, Nord Celébes,

13. IV 95 (53 mm).

Diese äthiopisch-orientalische Gattung erstreckt sich noch über

Celébes hinaus, bis Ternate. Von Celébes ist M. spasma schon
länger bekannt, allein genauere Fundorte wurden früher nicht
angegeben. Ich brachte sie von Gorontalo (Mus. Berl.), J e n t i n k
(NLM. V, 174 1883) hat sie von Amurang genannt, und (Cat. MPB.
XII, 170 1888) von Kema, das Dresdner Museum besitzt 3
Exemplare ebenfalls von Amurang in der Minahassa. Von Süd
Celébes scheint sie noch nicht gekommen zu sein. Ihre g a n z e
Verbreitung im Ostindischen Archipel von Hinterindien an ist noch
nicht bekannt.

Das S a r a s i n sche Weibchen von Nord Celébes hat einen

kleineren Vorderarm als D o b s o n im Allgemeinen angiebt (Cat.
1878, 158), 58 mm (2.3 inch.) gegen 53, und die drei anderen
Exemplare aus der Minahassa im Dresdner Museum messen auch
kaum mehr, die von Sumátra aber 57–59 mm.
[Inhalt]
 Vespertilionidae

[Inhalt]

17. Vesperus pachypus (Temm.)

mas,a.in Spiritus, Kema, Minahassa, Nord Celébes, 93 (26

mm).
fem.,
b–m.dsgl. (25–27 mm).

Diese von Vorderindien bis zu den Philippinen und Java verbreitete

Art wurde zuerst von J e n t i n k (NLM. V, 175 1883, XI, 30 1888 u.
Cat. MPB. XII, 176 1888) von Amurang, Nord Celébes, aufgeführt,
mir liegt sie auch von der Insel Saleyer, im Süden, vor, während sie
von Bórneo noch nicht nachgewiesen ist.

P e t e r s (Mb. Ak. Berl. 1872, 705) hat von mir aus Luzon
mitgebrachte kleinere als meyeri abgetrennt, was D o b s o n (Cat.
1878, 208) jedoch nicht anerkannte. Ein mir von Mindanao
vorliegendes Exemplar ist ebenfalls in allen Dimensionen kleiner
(Vorderarm 24 mm), was aber besonders, gerade wie bei denen von
Luzon, „in Bezug auf den Kopf und Fuss auffallend“ ist (vgl.
P e t e r s ’ Maasse mit D o b s o n s ll. cc.). D o b s o n giebt für
pachypus die Vorderarmlänge auf 28 mm (1.1 inch.) an, drei
Saleyer-Exemplare messen 26–26.5. Ich möchte daher die
Identificirung D o b s o n s nicht ohne Weiteres als berechtigt
ansehen. [13]
[Inhalt]

18. Vesperugo petersi n. sp.

Tafel IV Fig. 2 (​2⁄1 n. Gr.)

V. brunneus (Ridgway III, 5), alis nigris; auriculis parvis,

triangularibus, apice rotundatis, trago margine interno fere recto,
externo convexo, apice subacuto; alis malleolis affixis; cauda
apice extremo prominente; incisivo primo superiore bifido,
secundo paene ejusdem longitudinis, praemolari secundo 1
superiore bene evoluto et extrinsecus visibili; incisivorum
inferiorum tractu eodem quo mandibulae margines; pene
ossiculo armato.
Long. tot c. 90, antibr. 36.5 mm.
Habitatio: Celébes.
mas,a.
in Spiritus, Minahassa, Nord Celébes.

O b e r s e i t e vandykebraun, die Haare einfarbig, U n t e r s e i t e

etwas mehr rostfarben, die Haare an der Basis grau bis schwärzlich,
Fell sammetartig. F l u g h ä u t e schwarz, an der dorsalen und
ventralen Oberfläche nackt bis auf ein Dreieck zwischen den
Körperseiten und der proximalen Hälfte des Femur, und bis auf ein
ebensolches an der Schwanzbasis; am Knöchel angewachsen; die
Schenkelflughaut schliesst den Schwanz bis zum äussersten Glied
ein; S p o r n mit einem schmalen Hautläppchen am mittleren Drittel.
O h r e n innen sehr sparsam hellbräunlich behaart, Innen- und
Aussenrand convex, crus helicis lappig, Antitragus gut abgesetzt, 2
mm hinter dem Mundwinkel endend, Spitze abgerundet, T r a g u s
ziemlich gleich breit in seinem ganzen Verlauf, am inneren Rande
wenig concav, fast gerade, am äusseren convex, Zacken
angedeutet, Spitze schwach abgerundet. S c h n a u z e n d r ü s e n
gut entwickelt, schwach behaart, Nasenlöcher nach aussen offen,
innen vorgewölbt, dazwischen nicht vertieft.

i 1 sup. zweispitzig, i 2 einspitzig und kaum kürzer als die hintere

Zacke von i 1, auch im Querschnitt an der Basis i 1 nicht sehr viel
nachstehend; p 2 sup. innenständig, spitz, ⅔ so gross wie p 1 und
von aussen sichtbar, p 1 ein wenig von c abgerückt und halb so lang;
i 1–3 inf. dreilappig und in der Richtung des Kiefers stehend, nicht
übereinander greifend, i 3 durch ein kleines Diastema von i 2
getrennt und ein wenig quer gerückt (die linken unteren Incisiven
etwas verletzt); p 2 inf. mit der Spitze etwas nach aussen ausladend,
durch ein kleines Diastema von c getrennt und ⅔ so gross wie p 1,
dieser ⅔ so lang wie c.

Ein spitzer, 2.5 mm langer Stützknochen in der Ruthenspitze.

Maasse:

Kopf 17 mm
Körper 32
,,
Ohr 11 × 6.8
,,
Vorderer Ohrrand 7
,,
Tragus 5×2
,,
Humerus 24
,,
Vorderarm 36.5
,,
Dig. 1 mit Kralle 9
,,
 2 (33 + 4?) 37
,, ,,
64.5
3 (35 + 12.5 + 10 + 7)
,, ,,
4 (34 + 12 + 8) 54
,, ,,
5 (32 + 7.5 + 5) 44.5
,, ,,
Femur 12
,,
Unterschenkel 15.5
,,
Fuss mit Krallen 8.5
,,
Sporn 18
,,
Schwanz 41
,,
Penis 9 2
,,

Ich widme diese Art dem Andenken meines hochverehrten Lehrers

und Freundes W i l h e l m P e t e r s , der sich bekanntlich um die
Förderung der Kenntnisse der Fledermäuse grosse Verdienste
erworben hat. So sagt D o b s o n (Cat. 1878 p. XXXV): „P e t e r s , to
whom we owe the first attempt to arrange scientifically many of the
genera of Chiroptera“, und: „At Berlin, through the great liberality of
Prof. P e t e r s , I had the privilege of inspecting the beautifully
executed series of unpublished plates representing the species of
Chiroptera in the collection of the Royal Zoological Museum.“ Leider
hat P e t e r s die von ihm vorbereitete Monographie dieser Ordnung
1883 unvollendet hinterlassen, allein ihre Veröffentlichung, auf den