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in developing biopharmaceuticals
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Houde (Editor)
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BIOPHYSICAL CHARACTERIZATION OF
PROTEINS IN DEVELOPING
BIOPHARMACEUTICALS
SECOND EDITION
Edited by
DAMIAN J. HOUDE
Biomolecular Discovery, Relay Therapeutics,
Cambridge, MA, United States
STEVEN A. BERKOWITZ
Consultant, Sudbury, MA, United States
Elsevier
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Contributors
Yves Aubin Centre for Biologics, Regulatory David A. Keire Food and Drug Administration,
Research Division, Evaluation, Biologics and St. Louis, MO, United States
Genetic Therapies Directorate, Health Products Francis Kinderman Amgen, Thousand Oaks,
and Food Branch, Health Canada, Ottawa, ON, California, United States
Canada
Lee Makowski Department of Bioengineering
Steven A. Berkowitz Consultant, Sudbury, MA, and Department of Chemistry and Chemical
United States Biology, Northeastern University, Boston, MA,
George M. Bou-Assaf Analytical Development, United States
Biogen, Cambridge, MA, United States John P. Marino National Institute of Standards
Mark L. Brader Drug Product Analytical and Technology, Institute for Bioscience and
Development, Moderna, Cambridge, MA, Biotechnology Research, Rockville, MD, United
United States States
Richard K. Burdick Burdick Statistical Con- Alan G. Marshall Ion Cyclotron Resonance
sulting, LLC, Colorado Springs, CO, United Program, National High Magnetic Field
States Laboratory, Florida State University, Talla-
John F. Carpenter Department of Pharmaceut- hassee, FL, United States; Department of
ical Sciences, Center for Pharmaceutical Bio- Chemistry & Biochemistry, Florida State
technology, University of Colorado Anschutz University, Tallahassee, FL, United States
Medical Campus, Aurora, CO, United States A.J. Miles Institute of Structural and Molecular
Stephen J. Demarest Eli Lilly Biotechnology Biology, Birkbeck College, University of Lon-
Center, San Diego, CA, United States don, London, United Kingdom
Ertan Eryilmaz Takeda, Cambridge, Massachu- John S. Philo Alliance Protein Laboratories, San
setts, United States Diego, CA, United States
Verna Frasca Field Applications Manager, Mal- Angelika Reichel Coriolis Pharma, Munich,
vern Panalytical, Northampton, MA, United Germany
States Deniz B. Temel Bristol-Myers Squibb, Devens,
Darron L. Freedberg Structural Biology Section, Massachusetts, United States
Laboratory of Bacterial Polysaccharides, Silver B.A. Wallace Institute of Structural and Molec-
Spring, MD, United States ular Biology, Birkbeck College, University of
John P. Gabrielson Elion Labs, A Division of London, London, United Kingdom
KBI Biopharma, Inc., Louisville, CO, United Daniel Weinbuch Coriolis Pharma, Munich,
States Germany
Andrea Hawe Coriolis Pharma, Munich, William F. Weiss IV Bioproduct Research and
Germany Development, Lilly Research Laboratories, Eli
Damian J. Houde Biomolecular Discovery, Lilly and Company, Indianapolis, IN, United
Relay Therapeutics, Cambridge, MA, United States
States Sarah Zölls Coriolis Pharma, Munich, Germany
xi
Prefaces for the second edition
Critical to the development of any scientific developments and to the realiza-

successful therapeutic drug is our ability to tion that there was room for improvements.
identify and manufacture the drug such As a result, in writing this second edition
that its beneficial therapeutic effect can be we have undertaken the job of updating old
safely delivered to the patient. In the case of information, correcting mistakes, improving
protein biopharmaceuticals, these large, clarity and the introduction of new topics
heterogeneous (complex) and marginally that were not covered in the first edition.
stable molecules are often very sensitive to Therefore, we gathered our co-authors once
their micro-environment. This makes the again, invited a few new ones, and tasked
process of developing and manufacturing a ourselves with the goal to achieve these
protein therapeutic extremely challenging. objectives. In so doing, all original chapters
Throughout this entire process a protein bio- have been updated, corrected and
pharmaceutical must maintain its complex enhanced, while new chapters have been
and delicate structure (or conformation) to added.
realize its beneficial therapeutic attributes, Globally, the format of the book has
while avoiding the potential harmful effects remained the same, consisting of three sec-
in failing to achieve this goal. tions. Section I, which deals with the
When we set out to write the first edition complexity of proteins and the relevance of
of this book, our goal was to provide a biophysical methods in the biopharmaceuti-
general resource that specifically dealt with cal industry. It has for the most part been
the many challenges associated with the altered to remove errors and achieve clarity.
testing and characterization of the higher Section II, which discusses the biophysical
order structure and biophysical properties of tools and techniques most commonly used in
protein biopharmaceuticals from a practical the biopharmaceutical industry to charac-
point of view to support its safety and terize protein therapeutic molecules has
beneficial therapeutic activity. As stated in similarly been altered, but has also been
the book’s first preface we wanted to keep enhanced by the addition of a new chapter
the reader focused on obtaining a pragmatic (Chapter 14) dedicated to the area of chro-
understanding and knowledge of the utility matography and electrophoresis. The tools in
of biophysical tools and how they are used to this chapter, which we did not cover in the
meet these challenges by understanding first edition of the book (with the exception of
what information can realistically be extrac- size-exclusion chromatography), are typically
ted from these tools. While we felt we had not thought of or classified as biophysical
initially achieved our goal, the progression of tools. Nevertheless, an important objective in
time inevitably led to better and improved adding this chapter is to bring more attention
xiii
xiv PREFACES FOR THE SECOND EDITION
to their unrealized linkage as effective bio- Finally, we would like to point out that
physical characterization tools without get- in writing this second edition we have
ting too deep into the details of their inner made a particular effort, wherever possible,
workings (which are extensively covered in to better link and cross-reference informa-
many excellent books and review articles that tion in each chapter to bring more cohesion
are solely dedicated to these two enormously to the book as appose to just providing
important techniques). the reader with a collection of isolated
Overall, however, Section III of the book chapters. We think his cohesion is in partic-
has experienced the most significant change ular made apparent by the four additional
and expansion via the addition of four new chapters in Section III (described above).
chapters that cover the following: In the end, we and our coauthors hope we
have further enhanced the initial objective of
• Chapter 15, which deals with the biophysical
characterization of complex biopharmaceuticals; the first edition of the book, of enlightening
the reader to the challenges, tools and inner
• Chapter 16, which deals with the rigor of
workings of the task associated with the
statistical analysis;
biophysical characterization of protein bio-
• Chapter 17, which deals with biopharmaceu-
pharmaceuticals. An integral part of today’s
tical developability;
modern and challenging world of devel-
• Chapter 18, which deals with technical deci-
oping lifesaving drugs.
sion making.
List of abbreviations and symbols
(T)RPS (Tunable) resistive pulse sensing

3D Three dimensional
A22 or B2 Second viral coefficient
AAV Adeno-associated virus
AC Alternating current
AC-SINS Affinity-capture self-interaction nanoparticle spectroscopy
AFFF-MALLS Asymmetric field flow fractionation with multi-angle laser light scattering
ACN Acetonitrile
ACS Ammonium camphor sulfonate
ADC or ADCs Analog to digital converter or Antibody drug conjugate(s)
ADCC Antibody dependent cell-mediated cytotoxicity
AF4 Asymmetric flow field flow fractionation
AFM Atomic force microscopy
API Active pharmaceutical ingredient
APR Aggregation prone regions
AQL Acceptable quality level
ASTM American society for testing and materials
ATP Analytical target profile
ATR Attenuated total reflectance
AUC Analytical ultracentrifugation
BiAb or bsAb Bispecific antibody
BLA Biological license application
BMI Backgrounded membrane imaging
BSA Bovine serum albumin
CA Capsid protein
CAD Collision-activated dissociation
CCD Charge-coupled device
CD Circular dichroism
CDER Center for drug evaluation and research
CDR Complementarity-determining region
CEX Cation-exchange chromatography
cGMP Current good manufacturing practices
CH Immunoglobulin gamma heavy chain constant domain
CH1 or CH1 Immunoglobulin gamma heavy chain constant domain 1
CHO Chinese hamster ovary
CIC Cross-interaction chromatography
CID Collision induced dissociation
xv
xvi LIST OF ABBREVIATIONS AND SYMBOLS
cIEF Capillary isoelectric focusing

CIU Collision-induced unfolding
CL Immunoglobulin gamma light chain constant domain
CMC Manufacturing and Control
COSY Correlation spectroscopy
cP Centipose
CPL Circularly polarized light
CQA or CQAs Critical quality attribute(s)
CSA Camphor sulfonic acid
CZE Capillary (free) zone electrophoresis
D Deuterium or translational diffusion coefficient or electric dipole
DAC Deutscher arzneimittel-codex
DC Direct current
DI Developability index
DLS Dynamic light scattering
DoE Design of experiment
DNA Deoxyribonucleic acid
DOSY Diffusion ordered spectroscopy
DP Drug product
dPLIMSTEX Dilution PLIMSTEX
DRI Differential refractive index detector
DS Drug substance
DSA 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate
DSC Differential scanning calorimetry
DSF Differential scanning fluorimetry
DSLS Differential static light scattering
DSS 4,4-dimethyl-4-silpentane-1-sulfonic acid
DTT Dithiothreitol
ECD Electron capture dissociation or equivalent circular diameter
ECHOS Easy comparability of higher order structure
EDTA Ethylene diamine tetra-acetic acid
EM Electromagnetic radiation or electron microscopy
EMEA European Medicines Agency
ESD Equivalent sphere diameter
ESI Electrospray ionization
ESZ Electrical sensing zone
ET Electron tomography
ETD Electron transfer dissociation
EX1 H/D exchange mechanism in which the rate constant for protein folding/unfolding
is much slower than the rate constant for H/D exchange
EX2 H/D exchange mechanism in which the rate constant for protein folding/unfolding
is much faster than the rate constant for H/D exchange
Fab Immunoglobulin gamma fragment antigen binding
Fc Immunoglobulin gamma fragment crystallizable (constant region)
FcgRIIIa Immunoglobulin gamma Fc receptor RIIIa
FcRn Neonatal Fc receptor
FDA Food and Drug Administration
FFF Field flow fractionation
FID Free induction decay
LIST OF ABBREVIATIONS AND SYMBOLS xvii
FIX Blood clotting factor IX
FL Fluorescence
FT-ICR Fourier transform ion cyclotron resonance
FTIR or FT-IR Fourier transform infrared spectroscopy
fuc Fucose
FUV-CD Far ultraviolet circular dichroism
FVIII Blood clotting factor VIII
gal Galactose
GlcNAc N-acetylglucosamine
GLP Good laboratory practices
GMP Good manufacturing practices
H/DX-MS or HDX-MS Hydrogen/deuterium exchange mass spectrometry
HSA Human serum albumin
HCH Human growth hormone
HCl Hydrochloric acid
HCLF High concentration liquid formulation
HDC Hydrodynamic chromatography
HDX Hydrogen/deuterium exchange
HF5 Hollow fiber flow field flow fractionation
HGH Human growth hormone
hI Hydrophobicity index
HIC Hydrophobic interaction chromatography
HILIC Hydrophilic interaction chromatography
HMQC Heteronuclear multiple quantum coherence spectroscopy
HMW High molecular weight
HOS Higher-order structure
HPLC High performance liquid chromatography
HRR-DSC High ramp rate differential scanning calorimetry
HSQC Heteronuclear single quantum coherence spectroscopy
HT High tension
HX Hydrogen exchange
ICH International conference on harmonization of technical requirements for regis-
tration of pharmaceuticals for human use
icIEF Imaging capillary isoelectric focusing
IDP Intrinsically disordered protein
IDR Intrinsically disordered region
IEC Ion-exchange chromatography
IEF Capillary isoelectric focusing
IF Intrinsic fluorescence
IFN IFNb or IFNb1a Interferon-b-1a
IgG1 Immunoglobulin gamma 1 or immunoglobulin G1
ILP Integer linear programming
IM Ion mobility
ITC Isothermal titration calorimetry
IUP Intrinsically unstructured protein
IUR Intrinsically unstructured region
IV Intravenous injection
kD Diffusion Interaction Parameter
LC/MS Liquid chromatography/mass spectrometry
xviii LIST OF ABBREVIATIONS AND SYMBOLS
LMW Low molecular weight

LNPs lipid nanoparticles
LO Light obscuration
LOQ Limit of quantitation
LS Light scattering
mAb Monoclonal antibody
MALDI Matrix-assisted laser desorption/ionization
MALLS or MALS Multiangle laser light scattering
man Mannose
MD Molecular dynamics
MEM Maximum entropy method
MEMS Micro-Electro-Mechanical Systems
MFI Micro-flow imaging
MHz Megahertz
MRE or [M.R.E] Mean residue ellipticity
mRNA Messenger ribonucleic acid
MS Mass spectrometry
MS/MS Mass spectrometry/mass spectrometry or tandem mass spectrometry
MW Molecular weight
NEM N-ethylmalemide
NIBS Noninvasive back scattering technique
nIEF Native isolelectric focusing
NIST National Institute of Science and Technology
NMR Nuclear magnetic resonance or nuclear magnetic resonance spectroscopy
NNLS Nonnegative least squares
NOE Nuclear Overhauser Effect
NOESY Nuclear Overhauser Effect spectroscopy
NTA Nanoparticle tracking analysis
OCD Oriented circular dichroism
OD Optical density
OQ Operation qualification
OS-GAGs Oversulfated glycosaminoglyclans
PBS Phosphate buffered saline
PCA Principal component analysis
PDA Photodiode-array
PDB Protein data bank
PDI Polydispersity index
PEG Polyethylene glycol
PEM Photoelastic modulator
PFG Pulsed field gradient
PFGE Pulsed field gradient echo
Phe Phenylalanine
pI Isoelectric point
PK Pharmacokinetic
PL Path length
PLIMSTEX Proteineligand interactions by mass spectrometry, titration, and H/D exchange
PMT Photomultiplier tube
PPC Procedure Performance Criterion
PPI Protein-protein interactions
LIST OF ABBREVIATIONS AND SYMBOLS xix
PPQ Procedure Performance Qualification
PQ Performance qualification
Pr Probability
PTM or PTMs Posttranslational modification(s)
QA Quality Analysis
QbD Quality by design
QToF Quadrupole time of flight
QTPP Quality target product profile
RDCs Residual dipolar couplings
RF Radio-frequency
rhGM-CSF Recombinant human granulocyte colony stimulating factor
rhuEPO Recombinant human erythropoietin
RI Refractive index
rmAb Recombinant monoclonal antibody
RMM Resonant mass measurement
RP-HPLC, RPLC or rpLC Reversed-phase high performance liquid chromatography or reversed-phase liquid
chromatography
RPS Resistive pulse sensing
RS Reference standard
RT Room temperature or retention time
S or s Standard deviation
S/N Signal to noise
SANS Small angle neutron scattering
SAP Spatial Aggregation Propensity
SAXS Small angle X-ray scattering
SC or SubQ Subcutaneous injection
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SE-AUC Sedimentation equilibrium analytical ultracentrifugation
SEC Size-exclusion chromatography
SE-HPLC or HP-SEC Size-exclusion high performance liquid chromatography or high-performance size-
exclusion chromatography
SEM Scanning electron microscopy
SFC Supercritical fluid chromatography
SIC Self-interaction chromatography
SIMCA Soft independent modeling of class analogy
SIMSTEX Self-association interactions by mass spectrometry, self-titration, and H/DX
SLS Static light scattering
SMP Submicron particles
SMR Suspended microchannel resonator
SPE Solid-phase extraction
SRCD Synchrotron radiation circular dichroism
STEM Scanning transmission electron microscopy
SUPREX Stability of unpurified proteins from rates of H/D exchange
SV-AUC Sedimentation velocity analytical ultracentrifugation
SVD Singular value decomposition
SVP Subvisible particles
T1 Longitudinal relaxation time constant
T2 Transverse relaxation time constant
TCEP-HCl Tris(2-carboxyethyl)phosphine hydrochloride
xx LIST OF ABBREVIATIONS AND SYMBOLS
TDA Taylor dispersion analysis

TEM Transmission electron microscopy
TIC Total ion current
TM-DSC or MT-DSC Temperature-modulated differential scanning calorimetry
TMU Target measurement uncertainty
TPP Target product profile
Try Tyrosine
TSP Trimethylsilyl propionate
Tyr Tryptophan
UPLC or UHPLC Ultrahigh performance liquid chromatography or ultra-performance liquid
chromatography
USP United States Pharmacopeia
UV Ultraviolet light
UVeVIS Ultravioletevisible spectroscopy
VH Immunoglobulin gamma heavy chain variable domain
VIS Visible light
VL Immunoglobulin gamma light chain variable domain
VLP or VLPs Virus-like particle(s)
WAXS Wide angle X-ray scattering
WCX Weak-cation exchange chromatography
WSD Weighted spectral difference
XIC Extracted ion chromatogram
Z Net charge
C H A P T E R
1
The complexity of protein structure
and the challenges it poses in
developing biopharmaceuticals
Steven A. Berkowitza, Damian J. Houdeb
a
Consultant, Sudbury, MA, United States; bBiomolecular Discovery, Relay Therapeutics,
Cambridge, MA, United States
1.1 The basics of protein higher order structure (HOS)

Proteins are an important class of large biological molecules that are classified more gener-
ally as macromolecules or polymers. However, given their biological origin, these unique
molecules are often referred to as biomacromolecules or biopolymers. They are truly com-
plex, particularly when compared to synthetic (man-made) polymers and even other types
of biopolymers, e.g., DNA. One of the main reasons for this complexity arises from their basic
building blocks, which in synthetic polymer chemistry are referred to as monomer units. In
the case of most synthetic polymers, the chemical composition consists typically of only one
type of monomer (although some synthetic polymers called copolymers or block-copolymers
are composed of two or possibly more different monomer units). Proteins made in nature via
a process called translation utilizing the genetic code are composed of not one, two, or even
three different monomer units, but rather are composed of as many as 20 different “natu-
rally” occurring monomer units called amino acids. These 20 amino acids (or proteinogenic
amino acids, which does not include the other know, but rare proteinogenic amino acids sele-
nocystine or pyrrolysine) are referred to as the standard amino acids. Although not all pro-
teins contain all 20 amino acids, most do. The presence of such a large diversity in chemical
composition, in virtually every protein, is a key element for their structural complexity, which
in turn gives rise to their diverse functionality. Indeed, this chemical complexity, coupled
with the large number of amino acid units or residues (N) present in proteins (that can number
in the thousands), and the uniqueness of the amino acids linear sequential arrangement
(which in protein chemistry is called the primary (1 ) structure, see Fig. 1.1A), enables a
Biophysical Characterization of Proteins in Developing

Biopharmaceuticals, Second Edition 3
https://doi.org/10.1016/B978-0-444-64173-1.00001-9 Copyright © 2020 Elsevier B.V. All rights reserved.
4 1. The complexity of protein structure and the challenges it poses in developing biopharmaceuticals
FIG. 1.1 (A) The linear sequential ordering of amino acids (represented by the rectangular black dashed boxes) in
a protein is referred to as its primary structure. The extreme left amino acid corresponds to the amino-terminus, while
the extreme right amino acid corresponds to the carboxyl-terminus end of the protein chain. The gray shaded area
corresponds to the peptide bonds that link all the amino acid units in a protein, yielding the polypeptide backbone (or
chain) indicated by the red (gray in print version) dotted rectangle. (B) An illustration of the planar structure of two
adjacent amide planes (each resulting from the double bond character, due to resonance, of the peptide bond shown
as black dashes), corresponding to the light blue (light gray in print version) shaded areas in (A), where the bottom
amide plane is formed from the peptide bond between the carboxyl group of amino acid 1 (containing R1) and the
amino group of amino acid 2 (containing R2) and the top amide plane is formed from the peptide bond formed
between the carboxyl group of amino acid 2 and the amino group of amino acid 3 (containing R3). Due to steric issues,
angular rotation around CaN (expressed by F, phi) and CCa (expressed by J, psi) bonds are limited. (C) A rep-
resentation of a common secondary structure, the a-helix. The small rectangle outlined in black dashes corresponds to
a small section of the helical arrangements of the amide planes, shown in (B).
staggering number of different possible proteins, 20N, to be made. Given the enormous array
of different proteins that can be made, the cell has exploited this diversity in protein structure
to create proteins to perform nearly every functional and structural role needed for its
existence.
I. Proteins and biophysical characterization in the biopharmaceutical industry

1.1 The basics of protein higher order structure (HOS) 5
In proteins, the amino acid units are linked together through a unique chemical bond
called the peptide bond, which is also referred to as the amide link, see Fig. 1.1A. The collection
of these peptide bonds in a given protein form a common element found in all proteins called
the polypeptide backbone or chain, see Fig. 1.1A. A unique feature of the peptide bond is the
planar structure that it forms between the carbonyl oxygen, carbon and the a-carbons (Ca
or alpha carbon) of one amino acid and the amide nitrogen, hydrogen and a-carbons of an
adjacent amino acid. The resulting planar feature of these linked atoms arises as a result of
the partial double bond character that exists between the carbonyl carbon (C) and the amide
nitrogen (N) atoms due to the presences of resonance structures, see Fig. 1.1B. This planar
structure and its attributes play an important role in a protein’s structure, as its presences
confines the polypeptide backbone to only certain configurations, via steric effects, which re-
stricts the angular range of bond rotation around the CaeN (expressed by F, phi) and C-Ca
(expressed by J, psi) bonds. These restrictions have been summarized in a 2-dimensional
graphical plot called the Ramachandran plot, developed by Ramachandran and others in
1963 [1]. Such a plot graphically shows how certain structural features of proteins can only
exist within a limited range of angles characterized by J and F, e.g., a-helix, see Fig. 1.1C.
These restrictions play an important role in the development of protein’s spatial structure
or higher order structure (HOS).
1.1.1 The levels of protein HOS

In developing protein biopharmaceuticals and in studying proteins in general, the most
important concept is “structure”. In the previous section, we briefly discussed the most basic
component of a protein’s structure, its linear sequence of amino acids, or primary structure.
However, the focus of this book is concerned with a protein’s three-dimensional (3D) or spatial
structure, also referred to as its conformation or HOS. Ultimately, when considering the struc-
tures of proteins, it is the HOS in concert with its primary structure (which also includes all
the primary chemical bond modifications that occur to its amino acid units, see Section 1.1.4)
that enables a protein to properly function or, as we will also discuss in latter sections,
malfunction.
In terms of protein HOS, there are three different levels that have been defined. These three
levels include: secondary (2 ), tertiary (3 ), and quaternary (4 ) structure, see Fig. 1.2. The first
two structural levels are concerned with a single polypeptide chain, while the latter is asso-
ciated with protein structures that involve the interaction of two or more polypeptide chains.
A protein’s 2 structure refers to the local folding patterns of a protein’s polypeptide chain, in
which the a-helix (see Fig. 1.2A), the b-sheet, turns, and random coils are the most prominent
resulting structural elements that are formed. These local folded elements can further partic-
ipate in higher levels of folding that involve an array of secondary structural elements that
give rise to the final 3D structure of a protein referred to as 3 structure of a protein; see
Fig. 1.2B. The summation of 2 , 3 and (if present) 4 structure, along with its entire 1 struc-
ture, is what gives a protein its unique structure, chemical and physical properties and
therefore its unique function. Indeed, it is this relationship between structure and function
that is the genesis of the protein “structure-function” concept, which states that a protein’s
structure determines its function.

FIG. 1.2 Illustration of the three levels of a protein’s HOS. (A) Representative secondary structural element, as
illustrated by a ribbon representative structure of an a-helix. (B) A cartoon representation of the folding of all the
secondary structural elements in a polypeptide chain, which gives rise to the polypeptide’s tertiary structure. (C) A
cartoon representation of the quaternary structure of a protein, which arises when the final protein structure involves
the association of more than one polypeptide chain to form the final folded protein structure (also see Fig. 1.3).
Although the folding and interactions of the secondary structural elements can give rise to
an enormous array of different protein tertiary structures, each with unique properties and
functions, it’s not uncommon to find that the 3 structure of a protein often consists of one
or more commonly folded patterns called motifs, super-secondary structures, or complex folds
[2e4]. These commonly folded structures contain several folded secondary elements
involving only a portion of the entire polypeptide chain of a protein, which can blur some
of the distinction between a protein’s 2 and 3 structure. Hence, one might look at motifs,
super-secondary structures, or complex folds as “local 3 structure”, while referring to the
3 structure of the entire protein molecule as its “global 3 structure”.
Another structural element that further subclassifies the structural level of a protein
between what we call a protein’s 2 and 3 structure is the concept of domain [5,6]. Domains
are typically a much larger collection of folded structural elements than motifs, supersecon-
dary structures, or complex folds. In terms of the global structure of a protein, domains corre-
spond to one or more independent compact region of a protein’s polypeptide chain, as

FIG. 1.3 Different representations of the HOS of a monomeric IgG1 antibody. The two heavy chains are color-
coded in blue (light gray in print version) and gray, while the two light chains are both color-coded in red (dark
gray in print version). (A) A ribbon model of an IgG1 antibody (PDB: 1HZH). The black circle corresponds to the
variable domain on one of the IgG1 light chain (VL). (B) A simplified cartoon of the monomeric IgG1 antibody
indicating the various sections of individual domains present. The black lines linking the various interchain domains
correspond to areas where covalent linkages exist (disulfide bonds) between different polypeptide chains in the IgG1
molecule. The black circle corresponds to the same VL domain in the IgG1 molecule as shown in (A). (C) A space-
filling structural model of the monomeric IgG1 antibody. The black circled region again corresponds to the same
VL domain in the IgG1 antibody as shown in (A). (D) A linear depiction of a monomeric IgG1 structure showing all
the various covalent linkages (disulfide bonds) present in the IgG1 antibody. Those disulfide bonds present within
the same polypeptide chain are referred to as intrachain disulfide bonds, while those disulfide bonds that link two
different polypeptide chains are referred to as interchain disulfide bonds.
indicated by the black circles shown in Fig. 1.3AeC. Proteins containing two or more do-
mains are frequently referred to as multidomain proteins. In these proteins, the domains
are chemically linked by short sections of the polypeptide chain that are typically highly flex-
ible, called a “linker”, but nevertheless exist as stable and independent folded units. In certain
cases, common domain structures can also be found in proteins much like that observed with
motifs, super-secondary structures, or complex folds.
What is interesting about these folded elements is that there is a certain amount of change
in the 1 structure that can be tolerated while still arriving at, effectively, the same folded
structure. This observation explains the common presence of similar secondary, super-
secondary, and even domain structures seen in different proteins with different sequences.
Hence, the formation of these basic folding elements can display some level of discrepancy
in terms of the required or allowable amino acid sequence variations and still give rise to

the same functioning protein. This feature plays an important role in biological evolution, in
generating HOS building blocks, and in controlling and regulating groups of proteins that
perform very similar functions in different biochemical pathways [7e9]. Nevertheless, it is
important to mention that in proteins, there exist many sequence regions where even a slight
change, i.e., one amino acid change or a minor chemical modification (e.g., oxidation, deami-
dation), can significantly alter a protein’s structure and therefore its function [10,11].
For many proteins, however, the unique folded state of its polypeptide chain is not the last
step in attaining a final overall 3D structure. Many proteins are composed of more than one
polypeptide chains, which may be identical or nonidentical, giving these proteins an added
level of structural complexity, 4 structure; see Fig. 1.2C.
When referring to a protein’s 4 structure, a lack of clarity or confusion can unfortunately
arise. An example is illustrated in Fig. 1.3. In this figure, a monomeric intact IgG1 antibody is
shown. However, this protein could be referred to as a protein dimer (made of two identical
protein units) or a protein tetramer made of four separate polypeptide chains, which in this
case are chemically cross linked via covalent bonds called disulfide bonds (which is the most
common primary bond used in nature to cross-link parts of polypeptides). Such a choice of
descriptive words unfortunately can lead to some confusion. As a result, some care should be
taken when describing the basic structure of a protein. In the case of the 4 structure of IgG1
molecule, as shown in Fig. 1.3, the use of a tetramer in the context of its 4 structure would be
correct. However, in the context of a complete functioning unit (in its lowest complete form)
the molecule is a monomer.
1.1.2 Stabilizing the HOS of proteins

In all three levels of a protein’s HOS (i.e., 2 , 3 , and 4 ), various changes in the conforma-
tion of the polypeptide chain(s) occur as a protein folds to reach its final native structure.
These changes are typically accompanied by an increase in overall structural order, which
imparts a significant reduction in the protein’s entropy that by itself is highly unfavorable,
in terms of the overall free-energy change. However, as a protein folds, various weak nonco-
valent (secondary) bonds form via ionic, dipoles (hydrogen bonds), nonpolar (hydrophobic
effect), and van der Waals interactions. These weak bonds involve the interactions of
amino acid side chains, as well as elements of the polypeptide backbone, particularly the
amide hydrogen. While individually these interactions are weak, during the folding process
their large number and the cooperative way they form provide the necessary enthalpic and
entropic driving forces (release of structured water via the hydrophobic effect) to override the
large unfavorable decrease in entropy that occurs as a protein folds into its native (more or-
dered) conformation. The stabilization of the folded protein, however, is only marginal.
Comparing the level of stabilization against the average thermal energy content of a protein
molecule (which is equal to kT, where k ¼ Boltzmann constant and T ¼ temperature) and
the distribution of this energy, in terms of the amount of thermal energy per molecule, a variety
of these weak secondary bonds can be broken as a function of time. Such spatial and temporal
rupturing of these weak secondary bonds enables a protein to display dynamic structural prop-
erties in its conformation (sometimes referred to as protein breathing). This dynamic property
can play an important role in a protein’s function [12e15] and stability [16,17]. This dynamic
property, however, can also constitute a weakness for protein biopharmaceuticals, given the

wide range of stressful environments an average biopharmaceutical must endure during its
biosynthesis, purification, formulation, packaging/storage, patient handling, and its adminis-
tration. Hence, in searching for a good therapeutic biopharmaceutical, scientists look for mol-
ecules with high stability, such that the dynamic properties of the protein do not result in loss
of activity or adverse structural changes. Proteins that have such attributes are said to have
good developability properties.
In addition to weak secondary bonds, stabilization of the HOS of a protein can be achieved
through primary bonds formed between folded elements within a protein. As already
mentioned, the most common such bond is the disulfide bond, see Fig. 1.3D. Although the
number of disulfide bonds found in a given protein typically amounts to only a few such
bonds per protein molecule (and may not even exist within some proteins), they often play
important roles in a protein’s overall structure-function and stability [18]. Disulfide bonds
can occur both within a single polypeptide chain (where they are referred to as intrachain di-
sulfide bonds; see Fig. 1.3C) and between two different polypeptide chains in the same pro-
tein (where they are referred to as interchain disulfide bonds; see Fig. 1.3C). Disulfide bonds
also occur between two different protein molecules where they function to stabilize large
complex multiprotein supramolecular structures [19]. Unfortunately, however, the formation
of disulfide bonds can go astray leading to altered HOS structures or aggregates via disulfide
scrambling or exchange between other disulfide bonds or free cysteine residues in the same
protein or different proteins. These modes of protein degradation [20e27] are another reason
why the biopharmaceutical scientist need to constantly scrutinize the structure of the bio-
pharmaceutical during its development.
1.1.3 Dynamics properties of a Protein’s HOS

The HOS of virtually all proteins is primarily held together by a large array of relatively
weak bonds. In the context of a protein’s thermal energy content, these bonds can break
enabling various levels of fluctuations within a protein’s HOS that can span an enormous
time range, from 1015 s to tens of seconds and even longer [12,28]. Again, the fluctuations
in a protein’s conformation essentially occur because of the opening or breaking of various
weak secondary bonds. The extent of these fluctuations in terms of amplitude and location
is very dependent on many factors, e.g., environmental conditions, the strength of each sec-
ondary bond, the distribution of these bonds within the protein, as well as the distribution of
thermal energy within the protein. Variations in these (and other) factors will determine the
location of which secondary bonds will break in a protein’s HOS and therefore, the nature of
the conformational change(s) and the population of protein molecules in a specific conforma-
tion as a function of time. While these changes are for the most part contained to a region
where the secondary bond(s) break, changes might also extend to other areas of the protein,
via allosteric effects. Due to the random nature of the thermal energy fluctuations within a
protein, a range of different conformations and populations of different conformational states
will exist at any one time. For the most part, the extent of change in a protein’s HOS are typi-
cally not that large and are often reversible allowing the altered protein structure to return to
its more stable conformations.
Consequently, in solution proteins exist as an ensemble of different conformations, rather
than as a single fixed unique conformation. This ensemble is limited and controlled by the

interplay of the overall structure of the protein and its physicochemical environment. How-
ever, under appropriate conditions, involving some form of stress or subtle changes in a pro-
tein’s chemical structure, changes in conformation may cause a protein to display different
physicochemical properties. In the case of a protein biopharmaceutical, changes in its phys-
icochemical properties could alter the drug’s ability to bind with its therapeutic target or
enable it to bind to different materials it encounters, e.g., various container closure surfaces
[29e33]. Other possible adverse events include the formation of aggregates that are nonfunc-
tional and/or even more concerning, immunogenic [34e36]. It should be noted that the
formation of aggregates and their associated link to loss of protein function and/or immuno-
genicity corresponds to one of the most common forms of protein degradation that is closely
monitored in the biopharmaceutical industry.
1.1.4 Finer structural alteration of proteins

Once a protein is synthesized, or as it is being synthesized, additional primary structural
changes can occur in vivo. In most cases, these changes are due to additional enzymatic pro-
cessing reactions involving a multitude of potential chemical modifications to various amino
acids, as well as changes involving cleavage or cross-linking reactions. These reactions may or
may not play an important role in the normal function/activity of a protein, but rather may
represent alterations that play out to the determent of the cell or even the organism due to an
immunogenic response. Generally, most modifications are confined to the protein’s surface.
However, modifications can also occur to the protein’s interior due to the dynamic properties
of its structure (which exposes these buried internal areas) or during its synthesis when these
normally buried internal areas had not had a chance to properly fold. Such alterations can
lead to changes in the local or global HOS of the protein. In general, these modifications
are referred to as posttranslational modifications (PTMs). Principally, PTMs occur in vivo and
the number of different PTMs that a protein can experience is quite large [37]. In eukaryotes,
one of the more common (and biopharmaceutically relevant) PTMs is glycosylation. This
modification involves the enzymatic addition of carbohydrate (also called glycan or sugar)
units to a protein at specific asparagine (where they are called N-linked glycan) or serine
or threonine (where they are called O-linked glycan) amino acid [38]. While most PTMs occur
in vivo (inside the cell), PTMs can also occur in vitro (outside the cell). These latter PTMs,
however, typically represent forms of protein degradation that occur due to direct physical
or chemical interactions (e.g., oxidation, deamidation, glycation, etc.) and are also of great
concern in the biopharmaceutical industry as they are often linked to instability leading to
lose of drug activity and adverse effects [3,39e44].
1.2 The search for how proteins attain their correct

HOS: the protein folding problem
In the 1950s and 1960s, biophysical research led scientists to the realization that a protein’s
HOS is effectively dictated by its primary sequence. Christian Anfinsen was the key scientist
who formalized this idea, and in 1972 was awarded the Nobel Prize in chemistry for his con-
tributions [45]. In the scientific literature, this idea has been frequently referred to as the

1.2 The search for how proteins attain their correct HOS: the protein folding problem 11
“Anfinsen dogma” or the “thermodynamic hypothesis”. The folding path a protein takes to
achieve its correct functional HOS is intrinsically dictated by its 1 structure (which may also
include PTMs). How the folding process advances so efficiently, in combination with the way
a protein is synthesized in vivo, in the specific physicochemical environment within the cell,
has fascinated scientists for many years [46]. This fascination stems from the realization that
proteins achieve their correct HOS within a matter of milliseconds to seconds!
In the 1960s, Cyrus Levinthal prosed the following interesting and simple problem con-
cerning protein folding. For a protein consisting of 100 amino acids in an initially unfolded
state, how long would it take this protein to find, through a completely random process,
its appropriate native HOS given its physicochemical environment [28]? This problem is
nicely restated in the words of Amit Kessel and Nir Ben-Tal in their book “Introduction to
Proteins: Structure, Function and Motion” [47] as follows:
Assuming that the protein folding process involves the free sampling of all possible conformations of the
protein (i.e., of each residue independently), and that each residue has at least three states, then the folding of a
100-residue protein is excepted to sample 3100 ¼ 5 1047 conformations. Now if we assume that it takes a
protein 1 picosecond to sample a single conformation, then the time it takes to sample all possible confor-
mations in order to find the right one should be 3100 1012 s ¼ 5 1035 s ¼ 1.6 1028 years. This period of
time is about 1018 times longer than the age of the universe!!
This simple problem proposed by Levinthal is called “Levinthal’s Paradox” and was a sig-
nificant driving force for the generating what is called “the protein folding problem”. Clearly,
the nature of protein folding is nowhere as simple as starting with the completely synthesized
and unstructured (denatured or random coil) form of a protein, which is then allowed to un-
dergo a completely random sampling process of conformational space. Protein folding must
proceed via a process that is enormously more efficient, but how!!? Answers to this problem
appear to lie within the idea of a “funnel-shaped folding energy landscape” [48e52], see
Fig. 1.4, which might possibly take advantage of the way proteins are made in vivo along
with a concept of “divide and conquer”. In this process a protein proceeds to fold through
a hierarchy of subassembly units called a “foldon” [53,54]. These units can fold somewhat
independent of each other in parallel to form relatively local higher order structures that
can eventually collapse into the final native HOS of the protein.
In general, the funneling process of protein folding is likely not as simple as that portrayed
in Fig. 1.4A. Rather, it is expected to be more complex and treacherous, as indicated in
Fig. 1.4B. In the latter scenario, a folding protein could encounter conformational states
that are not as optimally folded as its native state and contain high activation energy barriers
that inhibit its search to find the most stable conformation. Hence, the protein in these states
would find itself trapped, due to the high energy of activation needed to transition the mis-
folded state back into a more unfolded state so it can find its more stable and native form.
Although these misfolded protein forms may be encountered at very low levels under normal
conditions, the situation could escalate under stressed conditions, such as forcing a cell to
produce a large quantity of one protein in a very short period. For such a situation, a higher
frequency of misfolded or metastable folded protein states could be encountered leaving the
biopharmaceutical scientist with a more difficult purification process that results in a lower
protein drug yield.

FIG. 1.4 A graphical view of the three-dimensional funnel-shaped energy landscape for protein folding. The top of
each funnel corresponds to the completely unfolded protein. The bottom of each funnel plot corresponds to the fully
folded protein molecule in its native state, which under closer scrutiny actually consists of a large array of slight
different energetically folded states (conformations) that differ in most cases by a small amount of free energy thus
enabling the native protein to exist in solution as an ensemble of different conformations. (A) A folding process free of
situations where it can be trapped in incomplete or partial folded state. (B) A folding process that enables partially
folded proteins to be potentially trapped due to the presence of smaller shaped folding funnels with relatively large
energy of activation that must be overcome in order to escape and find its final native state.
1.2.1 In vivo production of proteins: revisiting the protein folding problem

Another unique attribute of proteins is the complex manner with which they are made
in vivo. Protein synthesis involves a complex array of cellular machinery, the main compo-
nent of which is the ribosome. In vivo, proteins are synthesized from the N-terminus to
the C-terminus in a sequential manner at a rate of 50e300 amino acids/min [55,56]. The spe-
cific ordering and chemical coupling of the amino acids for a given protein is achieved by a
process called translation, which controls the protein synthesis process dictated by the genetic
coding information stored in a specific messenger RNA (m-RNA). As the nascent protein
chain is synthesized and exposed to the cell matrix, it can begin to fold. However, it should
be noted that the first 50e60 amino acids in the growing polypeptide are initially limited to
some extent in their ability to freely fold, due to the physical restrictions (steric hindrance) of
the environment within the ribosome [57]. This idea of concurrent, in vivo, protein synthesis
and folding are referred to as cotranslational protein folding [58] and likely plays an important
role in the folding of newly synthesized polypeptide.

1.2 The search for how proteins attain their correct HOS: the protein folding problem 13
The importance of cotranslational protein folding most likely arises because only the
growing polypeptide chain that has advanced beyond the ribosome tunnel will be able to
fully participate in the folding process. This allows only a portion of the growing protein
chain to fold without the interference from other parts of the protein that has either not
been synthesized or is located in the ribosome tunnel. As a result, this should improve the
efficiency of the sequential folding of the local higher order structural elements characterized
as foldon units to proceed in a more orderly manner. Such foldon units most likely corre-
spond to local HOS elements that are present in the final native protein. Nevertheless, as
these local higher order structural elements are formed, they must search out and undergo
higher levels of folding as the protein chain continues to grow. As a result, these various hi-
erarchy of folded structural elements are probably not arranged or packed optimally (as they
are in the protein’s final native state) until the entire protein is fully synthesized and release
from the ribosome. Once this happens, what remaining loose arrangement of folded (or
partially folded) structural elements that still exist must collapse into the final native structure
of the protein. This final consolation of folded or partially folded structural elements most
likely proceeds through the interactions of key amino acid side chains to make the final func-
tional protein (notwithstanding any additional changes in HOS resulting from PTMs and the
formation of quaternary structures).
Consequently, cotranslational protein folding constrains and to some extent guides the
overall protein folding process. By limiting the number of folding pathways (specifically
bad folding pathways, which would significantly increase the amount of time required to
find the correct native conformation) available to a protein, relative to the situation where
folding only begins once the protein is fully synthetized and release from the ribosome, could
make the role of cotranslational protein folding truly an important attribute in the successful
folding of a protein.
1.2.2 In vivo production of proteins: avoiding and eliminating folding errors

via the use of chaperones
In vivo, there are mechanisms involving other proteins, called chaperones, that help pro-
teins that are folding avoid the situation of being misfolded. This task is achieved via the
chaperone’s ability to assist a folding protein to avoid folding traps by participating in the
protein folding process through proteineprotein interactions [59e63]. In addition to chaper-
ones, there also exists in vivo cellular machinery whose function is to identify the presences of
misfolded proteins and eliminate them via proteolytic hardware existing within the cell [64].
However, these systems are not perfect, and failure to remove or prevent these erroneously
folded proteins from accumulating within the cell can alter the cell, causing adverse effects
that could eventually lead to its death. In the case of producing a protein biopharmaceutical,
once a misfolded protein is released into the cell culture media, it then becomes the problem
for the process scientist to develop appropriate purification strategies to remove the mis-
folded protein from the final protein drug product. If these erroneously folded proteins are
not removed, they could lead to adverse effects when the final drug product is administered
to a patient. Hence biophysical analysis of the biopharmaceutical’s HOS again becomes an
important activity in developing protein biopharmaceuticals with minimal levels of these
misfolded forms in the final drug product.

1.3 Surprises in the world of protein folding: intrinsically disordered or

unstructured proteins (an apparent challenge to the protein
StructureeFunction paradigm)
Within the past two decades, it has been realized that many proteins, especially in eukary-
otes or multicellular organism, exist within the cell with no defined HOS [28]. Rather, these
proteins appear to be disordered or unstructured, approaching what might be called a
random coil structure, anomalous to what is frequently seen with synthetic polymers or
denatured proteins. However, when these proteins interact with their target molecule(s)
they commonly appear to take on a level of organized HOS. Hence, this structural disorder
is transient in many cases and a disorder-to-order transition occurs during their functioning
(i.e., interacting with their binding target). Such behavior could play an important role in
allowing these proteins to bind to an array of different partner molecules by taking advantage
of the plasticity of their polypeptide chain’s flexibility [28]. This process is liable to be modu-
lated by other factors within the cell, which control and regulate the binding partners they
interact with. Indeed, the level of disordered proteins is higher in eukaryotes or multicellular
organism, in comparison to prokaryotes, where high levels of signaling and regulation is
required. This unique class of proteins has been referred to as intrinsically disordered pro-
teins (IDPs) or intrinsically unstructured proteins (IUPs) [65,66]. The existence of these
IDPs would appear to present a challenge to the paradigm of structureefunction discussed
earlier in this chapter.
With the realization of the existence of IDPs, many of the large random coil-like regions of
proteins consisting of 20e30 or more amino acids in length are now being referred to as
intrinsically disordered or unstructured regions (IDRs or IURs) [66]. These structural ele-
ments are commonly seen as linkers between ordered protein regions such as domains where
they are thought to also play important roles in providing protein flexibility, allowing proper
folding or to facilitate domainedomain interactions or domain binding to functioning bind-
ing targets. At present, IDPs have not made their way into the biopharmaceutical industry,
although it is probably only a matter of time until such a protein drug will appear.
1.4 Proteins and the biopharmaceutical industry: problems and

challenges
Although proteins can be chemically synthesized external to the cell, their high cost (which
is a function of protein size), as well as their overall complexity leads to poor economics for
building a viable commercial drug business via this approach. Over the years, however, sci-
entists have figured out how to get cells to produce significantly large amounts of a specific
protein, by manipulating their DNA via recombinant DNA technology. The development of
this capability was the key in enabling the biopharmaceutical drug industry to flourish. Over-
all this process of making protein biopharmaceutical differ greatly from the classical process
used to make simple organic drug molecules called pharmaceuticals, see Fig. 1.5. Cellular and
molecular biologist can now produce protein biopharmaceuticals at concentrations expressed
in the culture media volume as great as 10 g/L [67]. Nevertheless, the challenges of doing this

1.4 Proteins and the biopharmaceutical industry: problems and challenges 15
FIG. 1.5 A simple comparison illustrating differences in the process of making a pharmaceutical versus making a
biopharmaceutical: (A) Coarse outline of the sequential chemical reactions for making a pharmaceutical, using aspirin
as an example and (B) a coarse outline of the basic steps for making a biopharmaceutical, which consists of first
synthesizing a piece of DNA containing the correct nucleotide sequence code for making the desired bio-
pharmaceutical’s polypeptide chain(s), the insertion of this DNA into an initial small collection of cells (the microscale
factories for making the biopharmaceutical) using recombinant DNA technology, the large-scale growth of these cells
during which the cell’s internal protein synthesizing nano-machine (the ribosome, a complex cellular organelle
composed of many proteins and several pieces of RNA) are directed to synthesize the target biopharmaceutical,
illustrated here as either interferon beta-1a (IFNb) or a monoclonal antibody (mAb). Note that the space-filling
molecular models of aspirin, IFNb and mAb have all been displayed roughly on the same arbitrary scale to help
provide the reader with an approximate perspective on how they would relatively compare to each other on the basis
of size. The dashed circle highlighting part of the structure of IFNb corresponds to the carbohydrate-containing
portion of this biopharmaceutical that plays a dominant role in giving rise to its microheterogeneity shown in
chapter 2 in Figure 2.3 when coupled with other posttranslational modifications (PTMs). Reproduced with permission
from Berkowitz SA [116].
successfully are significant. Forcing a cell to produce unusually large amounts of a single
protein presents unique problems to the cell. Particularly in terms of making sure that all
the protein molecules are properly folded and have consistent physical, chemical, and biolog-
ical properties. Hence, to achieve this goal requires the constant and diligent monitoring and
characterization of the protein biopharmaceutical’s HOS.
The process of finding, developing, and obtaining regulatory approval of a protein
biopharmaceutical that is made using recombinant DNA technology proceeds through a
sequence of key activities or basic phases of activity that is outlined in Fig. 1.6. Success of

FIG. 1.6 A coarse overview of the basic areas and sequence of major activities involved in commercializing a
protein biopharmaceutical. The relative length of each block arrow is roughly associated with the length of time
typical spent at each stage, from research through commercialization. Overall, the cost in this process can easily be in
excess of one or more billion dollars and can require more than an decade to develop [68]. These numbers can vary
significantly from company to company and from drug to drug. As a result they are only approximate.
this process is highly dependent on biophysical characterization work associated with moni-
toring the consistency of the physicochemical properties of the protein drug, confirming the
absence of changes in the drug’s HOS (which might give rise to small unwanted subpopula-
tions of altered molecules), and in assessing the potential impact that PTMs might have on a
drug’s structure.
During the first part of this chapter we have dealt with the very basic properties of protein
structure. In the remaining sections, we will discuss how these properties are responsible for
many of the potential problems that are of great concern to a large range of biopharmaceu-
tical scientists. In addition, we will briefly look at some of the more novel types of protein
biopharmaceuticals that have and are being developed that further challenge the task of
biophysically characterizing these complex drugs.
1.4.1 Impact of PTMs on the HOS of protein biopharmaceuticals

The complex chemical composition of proteins, consisting of 20 chemically different natu-
rally occurring building blocks (i.e., amino acids) effectively empower the cell with the
needed components (chemistry set) to make the necessary array of proteins it needs to prop-
erly function. However, these amino acids also offer a range of chemically different targets
that can undergo chemical changes, via direct chemical reactions or through the participation
of various enzymatic reactions. The chemical changes that a protein biopharmaceutical can
incur offer opportunities to alter the HOS of these molecules, impacting the consistency of
manufacturing or worse, cause adverse events when administrated to a patient. As
mentioned in section 1.1.4, these chemical changes, whether they occur in vivo or in vitro,
are collectively referred to as PTMs. Many PTMs play roles in the biological function of a pro-
tein in vivo, while others are a result of normal degradation or aging. Hence, the idea that a
given protein exists as a single defined unique chemical entity is misleading. In fact, nearly all
protein biopharmaceuticals exist as a collection of highly similar variant forms. The range of
these variants and their amounts in the final biopharmaceutical drug product is determined
by the nature of the cell-line used, the cell culture conditions (e.g., raw materials and hold-
times within the bioreactor), the resolution properties of the purification process, as well as
our ability to detect and characterize them. The collection of highly similar proteins, variant
forms or proteoforms [69] of effectively the same protein that characterize a biopharmaceutical

is referred to as microheterogeneity and is a unique property of these drugs. In making a
protein biopharmaceutical, the attributes of microheterogeneity need to be carefully charac-
terized, measured and controlled. In so doing, microheterogeneity becomes a fingerprint or
a signature of the protein drug that is linked to its therapeutic behavior. However, it should
be noted that manufacturers of biopharmaceuticals cannot “exactly” duplicate this finger-
print or signature microheterogeneity on a lot-to-lot basis. Nevertheless, within the concept
of consistency of manufacturing, microheterogeneity needs to be contained to within an
established and reasonable level of variation that is bounded by the biopharmaceutical’s
specifications. The task of establishing this level of variation permitted in a bio-
pharmaceutical’s microheterogeneity is a collaboration between the drug manufacturer and
regulatory agencies (who will eventually review and approve the drug). Although, in the
end it is the regulators who have the final say on what is acceptable, in terms of necessary
specifications that defines this level of variation. These specifications are commonly associ-
ated with critical quality attributes (CQAs) and are attributes that are directly related to
the structural characteristics that define the chemical, physical, and biological properties of
the protein drug that impact the protein’s therapeutic activity.
As mentioned, PTMs occur both inside (in vivo) and outside (in vitro) of the cell. Key
factors that control in vivo PTMs include the following: cell line, culture media, and growth
conditions. In the case of in vitro PTMs, once the protein biopharmaceutical is excreted into
the culture media, the following are just a few key factors that can affect the protein drug
product: temperature, pH, contact surfaces, light, metal contamination, released enzymes
in the culture media or resulting from contamination, and sample handling (e.g., shaking,
freezing, and thawing) [70e75]. From beginning to end, there is a host of environmental chal-
lenges that the protein biopharmaceutical must endure without altering its primary structure
or HOS. In the case of the former, scientists in the biopharmaceutical industry have exten-
sively used mass spectrometry, MS, as a key analytical tool to detect and characterize
PTMs. This dependence on MS is primarily due to the change in mass that accompanies
nearly all chemical reactions and the high mass accuracy and resolution of most commercially
available MS instruments. However, it is worth noting that PTMs that involve isomerization
reactions and or isobaric mass transitions can occur, yielding no obvious or little mass change
and may require more sophisticated techniques to detect their presence [76e78]. In some
cases, these isobaric mass modifications can be chromatographically separated or fragmented
differentially via tandem MS [79]. In addition, although MS can detect, quantitate, and
localize PTMs within a protein, the impact of a PTM on a protein’s HOS is largely unknown.
Therefore, the application of biophysical analysis and characterization, as discussed in this
book, in combination with bioassays plays an important role in attempting to assess the
impact of PTMs in the biopharmaceutical industry.
1.4.2 Impact of changes in noncovalent interactions (secondary bonds) on the

HOS of protein biopharmaceuticals
Due to the high level with which a protein depends on an ensemble of weak secondary
bonds or interactions to maintain its HOS and dynamic nature of its conformation, a protein
can find itself potentially trapped in an altered conformation (metastable or intermediate
state) without any change in its primary structure. A protein is particularly vulnerable to

these HOS changes when placed under stress conditions. Under such stress conditions the
normally native protein can adopt a nonnative but energetically stable conformation (albeit
not as stable as its native conformation). When this partially unfolded protein state is accom-
panied with a relatively large kinetic energy activation barrier, when the stress is removed the
partially unfolded protein can find itself trapped; see Fig. 1.4B. In this situation, the protein
would encounter significant difficulty in returning to its more stable native state. As a result,
proteins can undergo an alteration in HOS “without” the need of requiring 1 structure
change. The ability to detect these types of HOS changes by MS would be very difficult since
the change in conformation would occur without any change in mass! Such changes in the
HOS of a protein can in terms of mass spectroscopy be considered silent HOS changes.
Thus, for the biopharmaceutical scientists to detect and quantify such changes a battery of
biophysical tools are often required.
1.4.3 A more detail discussion concerning protein biopharmaceutical

aggregations and its influence on HOS
Earlier in this chapter (Sections 1.1.2 and 1.1.3), it was mentioned that one of the most con-
cerning properties linked to biopharmaceutical proteins is their ability to self-associate and
form aggregates. While proteins can aggregate through many different mechanisms [80], in
general, protein aggregation can be crudely classified to arise from two basic properties of
a protein’s stability, its colloidal, and conformational stability. Aggregation resulting from
the attractive complex nature of the “normal” stable surface properties of a protein is a char-
acteristic associated with the protein’s colloidal properties and is related to its “colloidal
stability”. Aggregates formed via these properties are general referred to as “colloidal aggre-
gates”. However, due to the dynamic properties of proteins, these molecules can undergo a
range of fluctuations in its HOS, especially under stress conditions. Thus, changes in a pro-
tein’s conformational properties that expose buried (hydrophobic) chemical groups prone
to self-associate with other similar or different chemical groups on another protein molecule
are related to a protein’s “conformational stability”. Aggregates formed via these properties
are generally referred to as “conformational aggregates”. In some cases, aggregates that are
formed may not neatly arise from one form of stability or the other. Rather they might arise
through a hybrid combination of both [81,82].
In considering the unique self-associating properties of protein drugs, additional concerns
surface due to the presence of “macromolecular crowding” [83]. These concerns play a in two
areas which include: 1) the impact of the in vivo conditions of macromolecular crowding on
the self-association process, and 2) the impact of in vitro conditions required in developing
high protein biopharmaceutical concentration formulations [84,85]. In the former case, the
direct administration of a protein drug into the blood stream of a patient is thought to lead
to its rapid dilution, alleviating the adverse effect of a drug’s own high concentration.
However, the effect of macromolecular crowding resulting from the presence of other proteins
in the blood and lymph system along with the changes in the environmental solution condi-
tions, relative to the vialed-protein drug product can enhance self-association. As a result, better
methods for assessing these situations are needed [86,87], see chapter 15. In the case of devel-
oping high protein drug formulations for subcutaneous (SC) injection (which enables patient

convenience, avoiding the costly and inconvenient process of administrating large amounts
of protein drugs intravenously, IV), an additional question arises concerning the effects of
macromolecular crowding on protein drug self-association. This is because upon SC injection
a high protein concentration is deliberately created within the injected area that can remain at
a high concentration for a relatively long time due to the slow passive ability of the drug pro-
tein to find its way into the patient’s blood or lymph system in comparison to intravenous
injection (IV). In addition, the formulated SC delivered protein drug required for these
injections must also be stable and unaffected at these very high protein concentrations within
its container closure, e.g., vial or prefilled syringe, for several years [88], see chapter 15.
In general, the characterization and assessment of protein self-association is by no means
an easy task, especially at high protein concentrations. The bulk of the biophysical tools avail-
able to detect and characterize protein self-association have been developed for use on dilute
protein solutions, often referred to as ideal solution conditions. Here, the details of macromo-
lecular physical chemistry are simplified and much better understood. Tackling the solution
behavior of proteins at concentrations as high as several 100 mg/mL forces the biopharma-
ceutical scientist into experimental space where their ability to interpret acquired data is
extensively lacking, due to the poorly understood complexity of this situation and the
absence of a completely well-developed theory. As a result, conducting useful biophysical
characterization work is a real challenge resulting in biopharmaceutical scientists resorting
to either very empirical methodologies [89,90], or to the extrapolation of data from very
low concentrations to high concentrations [91e93] to make primitive and risky assessments,
again see Chapter 15 for further discussion on this topic.
1.4.4 The novelty of different classes of protein biopharmaceuticals that create

unique questions and challenges in characterizing and monitoring their HOS
Some protein biopharmaceuticals that have and are being developed contain unusual or
“unnatural” constructions and/or properties. In these cases, unique questions, problems,
and challenges arise concerning their physicochemical properties and HOS. Two of the
main types of “unusual” or “unnatural” drug candidates involve: (1) the covalent coupling
of a biopharmaceutical to another protein or chemical compound to create a fusion [78] or
conjugated (e.g., pegylated proteins [94,95], antibody drug conjugate, ADC [96], and XTEN
technology [97] protein biopharmaceutical); and (2) very large assembly of proteins such
as virus or virus-like particles (VLPs) or nanoparticle that serve as drug delivery systems
[98], see chapter 15. The following sections illustrate some of these novel biopharmaceuticals
that give rise to unique questions that bring into play the importance of biophysical measure-
ments concerning HOS.
1.4.4.1 Example 1: Fc fusion proteins

The fusion of an Fc (fragment crystallizable) part of an antibody (typically an IgG1
antibody) with that of another pharmaceutically relevant protein through recombinant tech-
nology, results in an Fc fusion protein. The reason for undertaking this fusion process is that
in an antibody, the Fc portion has been shown to be responsible for increasing the antibody’s
circulation time [99e101]. Thus, by expressing a pharmaceutically relevant protein fused with

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twenty-four hours was correspondingly decreased; while many a
tenement-house family spent Sunday in the country because for the
first time the head of the family could not use up his money in getting
drunk. The one all-important element in good citizenship in our
country is obedience to law, and nothing is more needed than the
resolute enforcement of law. This we gave.
There was no species of mendacity to which our opponents did
not resort in the effort to break us down in our purpose. For weeks
they eagerly repeated the tale that the saloons were as wide open as
ever; but they finally abandoned this when the counsel for the Liquor
Dealers’ Association admitted in open court, at the time when we
secured the conviction of thirty of his clients and thereby brought the
fight to an end, that over nine tenths of the liquor dealers had been
rendered bankrupt because we had stopped that illegal trade which
gave them the best portion of their revenue. They then took the line
that by devoting our attention to enforcing the liquor law we
permitted crime to increase. This, of course, offered a very congenial
field for newspapers like the World, which exploited it to the utmost;
all the more readily since the mere reiteration of the falsehood
tended to encourage criminals, and so to make it not a falsehood.
For a time the cry was not without influence, even with decent
people, especially if they belonged to the class of the timid rich; but it
simply wasn’t true, and so this bubble went down stream with the
others. For six or eight months the cry grew, first louder, then lower;
and then it died away. A commentary upon its accuracy was
furnished toward the end of our administration; for in February 1897,
the Judge who addressed the grand jury of the month was able to
congratulate them upon the fact that there was at that time less
crime in New York relatively to the population than ever before; and
this held true for our two years’ service.
In re-organizing the force the Board had to make, and did make,
more promotions, more appointments, and more dismissals than had
ever before been made in the same length of time. We were so
hampered by the law that we were not able to dismiss many of the
men whom we should have dismissed, but we did turn out 200 men
—more than four times as many as had ever been turned out in the
same length of time before; all of them being dismissed after formal
trial, and after having been given full opportunity to be heard in their
own defence. We appointed about 1700 men all told—again more
than four times as many as ever before; for we were allowed a large
increase of the police force by law. We made 130 promotions; more
than had been made in the six preceding years.
All this work was done in strictest accord with what we have grown
to speak of as the principles of civil service reform. In making
dismissals we paid heed merely to the man’s efficiency and past
record, refusing to consider outside pressure; under the old regime
no policeman with sufficient influence behind him was ever
dismissed, no matter what his offence. In making promotions we
took into account not only the man’s general record, his faithfulness,
industry and vigilance, but also his personal prowess as shown in
any special feat of daring, whether in the arresting of criminals or in
the saving of life—for the police service is military in character, and
we wished to encourage the military virtues. In making appointments
we found that it was practicable to employ a system of rigid
competitive examinations, which, as finally perfected, combined a
very severe physical examination with a mental examination such as
could be passed by any man who had attended one of our public
schools. Of course there was also a rigid investigation of character.
Theorists have often sneered at civil service reform as
“impracticable;” and I am very far from asserting that written
competitive examinations are always applicable, or that they may not
sometimes be merely stop-gaps, used only because they are better
than the methods of appointing through political endorsement; but
most certainly the system worked admirably in the Police
Department. We got the best lot of recruits for patrolmen that had
ever been obtained in the history of the force, and we did just as well
in our examinations for matrons and police surgeons. The uplifting of
the force was very noticeable, both physically and mentally. The best
men we got were those who had served for three years or so in the
Army or Navy. Next to these came the railroad men. One noticeable
feature of the work was that we greatly raised the proportion of
native-born, until, of the last hundred appointed, ninety-four per cent.
were Americans by birth. Not once in a hundred times did we know
the politics of the appointee, and we paid as little heed to this as to
their religion.
Another of our important tasks was seeing that the elections were
carried on honestly. Under the old Tammany rule the cheating was
gross and flagrant, and the police were often deliberately used to
facilitate fraudulent practices at the polls. This came about in part
from the very low character of the men put in as election officers. By
conducting a written examination of the latter, and supplementing
this by a careful inquiry into their character, in which we invited any
decent outsiders to assist, we very distinctly raised their calibre. To
show how necessary our examinations were, I may mention that
before each election held under us we were obliged to reject, for
moral or mental shortcomings, over a thousand of the men whom the
regular party organizations, exercising their legal rights, proposed as
election officers. We then merely had to make the police thoroughly
understand that their sole duty was to guarantee an honest election,
and that they would be punished with the utmost rigor if they
interfered with honest citizens on the one hand, or failed to prevent
fraud and violence on the other. The result was that the elections of
1895 and 1896 were by far the most honest and orderly ever held in
New York City.
There were a number of other ways in which we sought to reform
the police force, less important, and nevertheless very important. We
paid particular heed to putting a premium on specially meritorious
conduct, by awarding certificates of honorable mention, and medals,
where we were unable to promote. We introduced a system of pistol
practice by which, for the first time, the policemen were brought to a
reasonable standard of efficiency in handling their revolvers. The
Bertillion system for the identification of criminals was introduced. A
bicycle squad was organized with remarkable results, this squad
speedily becoming a kind of corps d’elite, whose individual members
distinguished themselves not only by their devotion to duty, but by
repeated exhibitions of remarkable daring and skill. One important bit
of reform was abolishing the tramp lodging-houses, which had
originally been started in the police stations, in a spirit of unwise
philanthropy. These tramp lodging-houses, not being properly
supervised, were mere nurseries for pauperism and crime, tramps
and loafers of every shade thronging to the city every winter to enjoy
their benefits. We abolished them, a municipal lodging-house being
substituted. Here all homeless wanderers were received, forced to
bathe, given night-clothes before going to bed, and made to work
next morning, and in addition they were so closely supervised that
habitual tramps and vagrants were speedily detected and
apprehended.
There was a striking increase in the honesty of the force, and
there was a like increase in its efficiency. When we took office it is
not too much to say that the great majority of the citizens of New
York were firmly convinced that no police force could be both honest
and efficient. They felt it to be part of the necessary order of things
that a policeman should be corrupt, and they were convinced that
the most efficient way of warring against certain forms of crime—
notably crimes against person and property—was by enlisting the
service of other criminals, and of purveyors of vice generally, giving
them immunity in return for their aid. Before we took power the
ordinary purveyor of vice was allowed to ply his or her trade
unmolested, partly in consideration of paying blackmail to the police,
partly in consideration of giving information about any criminal who
belonged to the unprotected classes. We at once broke up this
whole business of blackmail and protection, and made war upon all
criminals alike, instead of getting the assistance of half in warring on
the other half. Nevertheless, so great was the improvement in the
spirit of the force, that, although deprived of their former vicious
allies, they actually did better work than ever before against those
criminals who threatened life and property. Relatively to the
population, fewer crimes of violence occurred during our
administration of the Board than in any previous two years of the
city’s history in recent times; and the total number of arrests of
criminals increased, while the number of cases in which no arrest
followed the commission of crime decreased. The detective bureau
nearly doubled the number of arrests made compared with the year
before we took office; obtaining, moreover, 365 convictions of felons
and 215 convictions for misdemeanors, as against 269 and 105
respectively for the previous year. At the same time every attempt at
riot or disorder was summarily checked, and all gangs of violent
criminals brought into immediate subjection; while on the other hand
the immense mass meetings and political parades were handled with
such care that not a single case of clubbing of any innocent citizen
was reported.
The result of our labors was of value to the city, for we gave the
citizens better protection than they had ever before received, and at
the same time cut out the corruption which was eating away civic
morality. We showed conclusively that it was possible to combine
both honesty and efficiency in handling the police. We were attacked
with the most bitter animosity by every sensational newspaper and
every politician of the baser sort, not because of our shortcomings,
but because of what we did that was good. We enforced the laws as
they were on the statute books, we broke up blackmail, we kept
down the spirit of disorder, and repressed rascality, and we
administered the force with an eye single to the welfare of the city. In
doing this we encountered, as we had expected, the venemous
opposition of all men whose interest it was that corruption should
continue, or who were of such dull morality that they were not willing
to see honesty triumph at the cost of strife.
FOOTNOTES:
[14] Atlantic Monthly, September, 1897.
[15]My predecessor in the Presidency of the Police Board.
[16]The italics are my own.
IX
THE VICE-PRESIDENCY AND THE CAMPAIGN OF
1896[17]
The Vice-President is an officer unique in his character and

functions, or to speak more properly, in his want of functions while he
remains Vice-President, and in his possibility of at any moment
ceasing to be a functionless official and becoming the head of the
whole nation. There is no corresponding position in any
constitutional government. Perhaps the nearest analogue is the heir
apparent in a monarchy. Neither the French President nor the British
Prime Minister has a substitute, ready at any moment to take his
place, but exercising scarcely any authority until his place is taken.
The history of such an office is interesting, and the personality of the
incumbent for the time being may at any moment become of vast
importance.
The founders of our government—the men who did far more than
draw up the Declaration of Independence, for they put forth the
National Constitution—in many respects builded very wisely of set
purpose. In some cases they built wiser than they knew. In yet other
instances they failed entirely to achieve objects for which they had
endeavored to provide by a most elaborate and ingenious
governmental arrangement. They distrusted what would now be
called pure Democracy, and they dreaded what we would now call
party government.
Their distrust of Democracy induced them to construct the
electoral college for the choice of a President, the original idea being
that the people should elect their best and wisest men who in turn
should, untrammeled by outside pressure, elect a President. As a
matter of fact the functions of the electorate have now by time and
custom become of little more importance than those of so many
letter-carriers. They deliver the electoral votes of their states just as
a letter-carrier delivers his mail. But in the presidential contest this
year it may be we shall see a partial return to the ideals of the men
of 1789; for some of the electors on the Bryan-Sewall-Watson ticket
may exercise a choice between the vice-presidential candidates.
The distrust felt by the founders of the constitution for party
government took shape in the scheme to provide that the majority
party should have the foremost place, and the minority party the
second place, in the national executive. The man who received the
greatest number of electoral votes was made President, and the
man who received the second greatest number was made Vice-
President, on a theory somewhat akin to that by which certain
reformers hope to revolutionize our system of voting at the present
day. In the early days under the present constitution this system
resulted in the choice of Adams for President and of his anti-type
Jefferson as Vice-President, the combination being about as
incongruous as if we should now see McKinley President and either
Bryan or Watson Vice-President. Even in theory such an
arrangement is very bad, because under it the Vice-President might
readily be, and as a matter of fact was, a man utterly opposed to all
the principles to which the President was devoted, so that the
arrangement provided in the event of the death of the President, not
for a succession, but for a revolution. The system was very soon
dropped, and each party nominated its own candidates for both
positions. But it was many years before all the members of the
electoral college of one party felt obliged to cast the same votes for
both President and Vice-President, and consequently there was a
good deal of scrambling and shifting in taking the vote. When,
however, the parties had crystallized into Democratic and Whig, a
score of years after the disappearance of the Federalists, the system
of party voting also crystallized. Each party then as a rule nominated
one man for President and one for Vice-President, these being voted
for throughout the nation. This system in turn speedily produced
strange results, some of which remain to this day. There are and
must be in every party factions. The victorious faction may crush out
and destroy the others, or it may try to propitiate at least its most
formidable rival. In consequence, the custom grew of offering the
vice-presidency as a consolation prize, to be given in many cases to
the very men who were most bitterly opposed to the nomination of
the successful candidate for President. Sometimes this consolation
prize was awarded for geographical reasons, sometimes to bring into
the party men who on points of principle might split away because of
the principles of the presidential candidate himself, and at other
times it was awarded for merely factional reasons to some faction
which did not differ in the least from the dominant faction in matters
of principles, but had very decided views on the question of offices.
The presidency being all important, and the vice-presidency of
comparatively little note, the entire strength of the contending
factions is spent in the conflict over the first, and very often a man
who is most anxious to take the first place will not take the second,
preferring some other political position. It has thus frequently
happened that the two candidates have been totally dissimilar in
character and even in party principle, though both running on the
same ticket. Very odd results have followed in more than one
instance.
A striking illustration of the evils sometimes springing from this
system is afforded by what befell the Whigs after the election and
death of the elder Harrison. Translated into the terms of the politics
of continental Europe of to-day, Harrison’s adherents represented a
union between the right and the extreme left against the centre. That
is, the regular Whigs who formed the bulk of his supporters were
supplemented by a small body of extremists who in their political
principles were even more alien to the Whigs than were the bulk of
the regular Democrats, but who themselves hated these regular
Democrats with the peculiar ferocity so often felt by the extremist for
the man who goes far, but not quite far enough. In consequence, the
President represented Whig principles, the Vice-President
represented a rather extreme form of the very principles to which the
Whigs were most opposed. The result was that when Harrison died
the presidency fell into the hands of a man who had but a corporal’s
guard of supporters in the nation, and who proceeded to oppose all
the measures of the immense majority of those who elected him.
A somewhat similar instance was afforded in the case of Lincoln
and Johnson. Johnson was put on the ticket largely for geographical
reasons, and on the death of Lincoln he tried to reverse the policy of
the party which had put him in office. An instance of an entirely
different kind is afforded by Garfield and Arthur. The differences
between these two party leaders were mainly merely factional. Each
stood squarely on the platform of the party, and all the principles
advocated by one were advocated by the other; yet the death of
Garfield meant a complete overturn in the personnel of the upper
Republican officials, because Arthur had been nominated expressly
to placate the group of party leaders who most objected to the
nomination of Garfield. Arthur made a very good President, but the
bitterness caused by his succession to power nearly tore the party in
twain. It will be noted that most of these evils arose from the fact that
the Vice-President under ordinary circumstances possesses so little
real power. He presides over the Senate and he has in Washington a
position of marked social importance, but his political weight as Vice-
President is almost nil. There is always a chance that he may
become President. As this is only a chance it seems quite impossible
to persuade politicians to give it proper weight. This certainly does
not seem right. The Vice-President should, so far as possible,
represent the same views and principles which have secured the
nomination and election of the President, and he should be a man
standing well in the councils of the party, trusted by his fellow party
leaders, and able in the event of any accident to his chief to take up
the work of the latter just where it was left. The Republican party has
this year nominated such a man in the person of Mr. Hobart. But
nominations of this kind have by no means always been the rule of
recent years. No change of parties, for instance, could well produce
a greater revolution in policy than would have been produced at
almost any time during the last three years if Mr. Cleveland had died
and Mr. Stevenson had succeeded him.
One sure way to secure this desired result would undoubtedly be
to increase the power of the Vice-President. He should always be a
man who would be consulted by the President on every great party
question. It would be very well if he were given a seat in the Cabinet.
It might be well if, in addition to his vote in the Senate in the event of
a tie, he should be given a vote, on ordinary occasions, and
perchance on occasions a voice in the debates. A man of the
character of Mr. Hobart is sure to make his weight felt in an
administration, but the power of thus exercising influence should be
made official rather than personal.
The present contest offers a striking illustration of the way in which
the Vice-President ought and ought not to be nominated, and to
study this it is necessary to study not only the way in which the
different candidates were nominated, but at least in outline the
characters of the candidates themselves.
For the first time in many years, indeed for the first time since
parties have fairly crystallized along their present lines, there are
three parties running, two of which support the same presidential
candidate but different candidates for the vice-presidency. Each one
of these parties has carried several states during the last three or
four years. Each party has a right to count upon a number of
electoral votes as its own. Closely though the Democratic and
Populistic parties have now approximated in their principles as
enunciated in the platforms of Chicago and St. Louis, they yet do
differ on certain points, and neither would have any chance of
beating the Republicans without the help of the other. The result has
been a coalition, yet each party to the coalition has retained enough
of its jealous individuality to make it refuse to accept the candidate of
the other for the second position on the ticket.
The Republican party stands on a normal and healthy party
footing. It has enunciated a definite set of principles entirely in
accord with its past actions. It has nominated on this platform a
President and Vice-President, both of whom are thorough-going
believers in all the party principles set forth in the platform upon
which they stand. Mr. McKinley believes in sound finance,—that is, in
a currency based upon gold and as good as gold. So does Mr.
Hobart. Mr. McKinley believes in a protective tariff. So does Mr.
Hobart. Mr. McKinley believes in the only method of preserving
orderly liberty,—that is, in seeing that the laws are enforced at
whatever cost. So does Mr. Hobart. In short, Mr. Hobart stands for
precisely the same principles that are represented by Mr. McKinley.
He is a man of weight in the community, who has had wide
experience both in business and in politics. He is taking an active
part in the campaign, and he will be a power if elected to the vice-
presidency. All the elements which have rallied behind Mr. McKinley
are just as heartily behind Mr. Hobart. The two represent the same
forces, and they stand for a party with a coherent organization and a
definite purpose, to the carrying out of which they are equally
pledged.
It will be a matter of much importance to the nation that the next
Vice-President should stand for some settled policy. It is an
unhealthy thing to have the Vice-President and President
represented by principles so far apart that the succession of one to
the place of the other means a change as radical as any possible
party overturn. The straining and dislocation of our governmental
institutions was very great when Tyler succeeded Harrison and
Johnson succeeded Lincoln. In each case the majority of the party
that had won the victory felt that it had been treated with scandalous
treachery, for Tyler grew to be as repulsive to the Whigs as Polk
himself, and the Republicans could scarcely have hated Seymour
more than they hated Johnson. The Vice-President has a three-fold
relation. First to the administration; next as presiding officer in the
Senate, where he should be a man of dignity and force; and third in
his social position, for socially he ranks second to the President
alone. Mr. Morton was in every way an admirable Vice-President
under General Harrison, and had he succeeded to the presidential
chair there would have been no break in the great policies which
were being pushed forward by the administration. But during Mr.
Cleveland’s two incumbencies Messrs. Hendricks and Stevenson
have represented, not merely hostile factions, but principles and
interests from which he was sundered by a gulf quite as great as that
which divided him from his normal party foes. Mr. Sewall would make
a colorless Vice-President, and were he at any time to succeed Mr.
Bryan in the White House would travel Mr. Bryan’s path only with
extreme reluctance and under duress. Mr. Watson would be a more
startling, more attractive, and more dangerous figure, for if he got the
chance he would lash the nation with a whip of scorpions, while Mr.
Bryan would be content with the torture of ordinary thongs.
Finally, Mr. Hobart would typify as strongly as Mr. McKinley himself
what was best in the Republican party and in the nation, and would
stand as one of the known champions of his party on the very
questions at issue in the present election. He is a man whose advice
would be sought by all who are prominent in the administration. In
short, he would be the kind of man whom the electors are certain to
choose as Vice-President if they exercise their choice rationally.
The men who left the Republican party because of the nomination
of McKinley would have left it just as quickly if Hobart had been
nominated. They do not believe in sound finance, and though many
of the bolters object to anarchy and favor protection, they feel that in
this crisis their personal desires must be repressed and that they are
conscientiously bound to support the depreciated dollar even at the
cost of incidentally supporting the principles of a low tariff and the
doctrine that a mob should be allowed to do what it likes with
immunity. There are many advocates of clipped or depreciated
money who are rather sorry to see the demand for such currency
coupled with a demand for more lawlessness and an abandonment
by the government of the police functions which are the essential
attributes of civilization; but they have overcome their reluctance,
feeling that on the whole it is more important that the money of the
nation should be unsound than that its laws should be obeyed.
People who feel this way are just as much opposed to Mr. Hobart as
to Mr. McKinley. They object to the platform upon which the two men
stand, and they object as much to the character of one man as to the
character of the other. They are repelled by McKinley’s allegiance to
the cause of sound money, and find nothing to propitiate them in
Hobart’s uncompromisingly honest attitude on the same question.
There is no reason whatever why any voter who would wish to vote
against the one should favor the other, or vice versa.
When we cross the political line all this is changed. On the leading
issue of the campaign the entire triangle of candidates are a unit. Mr.
Bryan, the nominee for the presidency, and Messrs. Sewall and
Watson, the nominees for the vice-presidency, are almost equally
devoted adherents of the light-weight dollar and of a currency which
shall not force a man to repay what he has borrowed, and shall
punish the wrong-headed laborer, who expects to be paid his wages
in money worth something, as heavily as the business man or farmer
who is so immoral as to wish to pay his debts. All three are believers
in that old-world school of finance which appears under such protean
changes of policy, always desiring the increase of the circulating
medium, but differing as to the means, which in one age takes the
form of putting base metal in with the good, or of clipping the good,
and in another assumes the guise of fiat money, or the free coinage
of silver. On this currency question they are substantially alike,
agreeing (as one of their adherents picturesquely put it, in arguing in
favor of that form of abundant currency which has as its highest
exponent the money of the late Confederacy) that “the money which
was good enough for the soldiers of Washington is good enough for
us.” As a matter of fact the soldiers of Washington were not at all
grateful for the money which the loud-mouthed predecessors of Mr.
Bryan and his kind then thought “good enough” for them. The money
with which the veterans of Washington were paid was worth two
cents on the dollar, and as yet neither Mr. Bryan, Mr. Sewall, nor Mr.
Watson has advocated a two-cent copper dollar. Still, they are
striving toward this ideal, and in their advocacy of the fifty cent dollar
they are one.
But beyond this they begin to differ. Mr. Sewall distinctly sags
behind the leader of the spike team, Mr. Bryan, and still more
distinctly behind his rival, or running mate, or whatever one may
choose to call him, the Hon. Thomas Watson. There is far more
regard for the essential fitness of things in a ticket which contains Mr.
Bryan and Mr. Watson than one which contains Mr. Bryan and Mr.
Sewall. Mr. Watson is a man of Mr. Bryan’s type, only a little more
so. But Mr. Sewall is of a different type, and possesses many
attributes which must make association with him exceedingly painful,
not merely to Mr. Watson, but to Mr. Bryan himself. He is a well-to-do
man. Indeed in many communities he would be called a rich man.
He is a banker, a railroad man, a shipbuilder, and has been
successful in business. Now if Mr. Bryan and Mr. Watson really stand
for any principle it is hostility to this kind of success. Thrift, industry,
and business energy are qualities which are quite incompatible with
true Populistic feeling. Payment of debts, like the suppression of
riots, is abhorrent to the Populistic mind. Such conduct strikes the
Populist as immoral. Mr. Bryan made his appearance in Congress
with two colleagues elected on the same ticket, one of whom stated
to the present writer that no honest man ever earned $5000 a year;
that whoever got that amount stole it. Mr. Sewall has earned many
times $5000 a year. He is a prosperous capitalist. Populism never
prospers save where men are unprosperous, and your true Populist
is especially intolerant of business success. If a man is a successful
business man he at once calls him a plutocrat.
He makes only one exception. A miner or speculator in mines may
be many times a millionaire and yet remain in good standing in the
Populist party. The Populist has ineradicably fixed in his mind the
belief that silver is a cheap metal, and that silver money is, while not
fiat money, still a long step toward it. Silver is connected in his mind
with scaling down debts, the partial repudiation of obligations, and
other measures aimed at those odious moneyed tyrants who lend
money to persons who insist upon borrowing, or who have put their
ill-gotten gains in saving banks and kindred wicked institutions for
the encouragement of the vice of thrift. These pleasurable
associations quite outweigh, with the Populist, the fact that the silver
man himself is rich. He is even for the moment blind to the further
fact that these pro-silver men, like Senator Stewart, Governor
Altgeld, and their compeers, strenuously insist that the obligations to
themselves shall be liquidated in gold; indeed this particular
idiosyncrasy of the silver leaders is not much frowned upon by the
bulk of the Populists, because it has at least the merit of savoring
strongly of “doing” one’s creditors. Not even the fact that rich silver
mine owners may have earned their money honestly can outweigh
the other fact that they champion a species of currency which will
make most thrifty and honest men poorer, in the minds of the truly
logical Populist.
But Mr. Sewall has no fictitious advantage in the way of owing his
wealth to silver. He has made his money precisely as the most
loathed reprobate of Wall Street—or of New York, which the average
Populist regards as synonymous with Wall Street—has made his.
The average Populist does not draw fine distinctions. There are in
New York, as in other large cities, scoundrels of great wealth who
have made their money by means skilfully calculated to come just
outside the line of criminality. There are other men who have made
their money exactly as the successful miner or farmer makes his,—
that is, by the exercise of shrewdness, business daring, energy and
thrift. But the Populist draws no line of division between these two
classes. They have made money, and that is enough. One may have
built railroads and the other have wrecked them, but they are both
railroad men in his eyes, and that is all. One may have swindled his
creditors, and the other built up a bank which has been of
incalculable benefit to all who have had dealings with it, but to the
Populist they are both gold bugs, and as such noxious. Mr. Sewall is
the type of man the contemplation of which usually throws a Populist
orator into spasms. But it happens that he believes in free silver, just
as other very respectable men believe in spirit rapping, or the faith-
cure, or Buddhism, or pilgrimages to Lourdes, or the foot of a
graveyard rabbit. There are very able men and very lovely women
who believe in each or all of these, and there are a much larger
number who believe in free silver. Had they lived in the days of
Sparta they would have believed in free iron, iron coin being at that
time the cheapest circulating medium, the adoption of which would
give the greatest expansion of the currency. But they have been
dragged on by the slow procession of the centuries, and now they
only believe in free silver. It is a belief which is compatible with all the
domestic virtues, and even occasionally with very good capacities as
a public servant. Mr. Sewall doubtless stands as one of these men.
He can hardly be happy, planted firmly as he is, on the Chicago
platform. In the minds of most thrifty, hard-working men, who are
given to thinking at all about public questions, the free-silver plank is
very far from being the most rotten of the many rotten planks put
together with such perverted skill by the Chicago architects. A
platform which declares in favor of free and unlimited rioting and
which has the same strenuous objection to the exercise of the police
power by the general government that is felt in the circles presided
over by Herr Most, Eugene V. Debs, and all the people whose
pictures appear in the detective bureaus of our great cities, cannot
appeal to persons who have gone beyond the unpolished-stone
period of civilization.
The men who object to what they style “government by injunction”
are, as regards the essential principles of government, in hearty
sympathy with their remote skin-clad ancestors who lived in caves,
fought one another with stone-headed axes, and ate the mammoth
and woolly rhinoceros. They are interesting as representing a
geological survival, but they are dangerous whenever there is the
least chance of their making the principles of this ages-buried past
living factors in our present life. They are not in sympathy with men
of good minds and sound civic morality. It is not a nice thing to wish
to pay one’s debts in coins worth fifty cents on the dollar, but it is a
much less nice thing to wish to plunge one’s country into anarchy by
providing that the law shall only protect the lawless and frown
scornfully on the law-abiding. There is a good deal of mushy
sentiment in the world, and there are always a certain number of
people whose minds are weak and whose emotions are strong and
who effervesce with sympathy toward any man who does wrong, and
with indignation against any man who chastises the criminal for
having done wrong. These emotionalists, moreover, are always
reinforced by that large body of men who themselves wish to do
wrong, and who are not sentimental at all, but, on the contrary, very
practical. It is rarely that these two classes control a great political
party, but at Chicago this became an accomplished fact.
Furthermore, the Chicago convention attacked the Supreme
Court. Again this represents a species of atavism,—that is, of
recurrence to the ways of thought of remote barbarian ancestors.
Savages do not like an independent and upright judiciary. They want
the judge to decide their way, and if he does not, they want to
behead him. The Populists experience much the same emotions
when they realize that the judiciary stands between them and
plunder.
Now on all these points Mr. Sewall can hardly feel complete
sympathy with his temporary allies. He is very anxious that the
Populists shall vote for him for Vice-President, and of course he feels
a kindly emotion toward those who do intend to vote for him. He
would doubtless pardon much heresy of political belief in any
member of the electoral college who feels that Sewall is his friend,
not Watson,—Codlin, not Short. He has, of course, a vein of the
erratic in his character, or otherwise he would not be in such
company at all, and would have no quality that would recommend
him to them. But on the whole his sympathies must lie with the man
who saves money rather than with the man who proposes to take
away the money when it has been saved, and with the policeman
who arrests a violent criminal rather than with the criminal. Such
sympathy puts him at a disadvantage in the Populist camp. He is
loud in his professions of belief in the remarkable series of principles
for which he is supposed to stand, but his protestations ring rather
hollow. The average supporter of Bryan doubtless intends to support
Sewall, for he thinks him an unimportant tail to the Bryan kite. But,
though unimportant, he regards him with a slight feeling of irritation,
as being at the best a rather ludicrous contrast to the rest of the kite.
He contributes no element of strength to the Bryan ticket, for other
men who work hard and wish to enjoy the fruits of their toil simply
regard him as a renegade, and the average Populist, or Populistic
Democrat, does not like him, and accepts him simply because he
fears not doing so may jeopardize Bryan’s chances. He is in the
uncomfortable position always held by the respectable theorist who
gets caught in a revolutionary movement and has to wedge
nervously up into the front rank with the gentlemen who are not
troubled by any of his scruples, and who really do think that it is all
very fine and glorious. In fact Mr. Sewall is much the least
picturesque and the least appropriate figure on the platform or
platforms upon which Mr. Bryan is standing.
Mr. Watson, whose enemies now call him a Georgia cracker, is in
reality a far more suitable companion for Mr. Bryan in such a contest.
It must be said, however, that if virtue always received its reward Mr.
Watson and not Mr. Bryan would stand at the head of the ticket. In
the language of mathematicians Mr. Watson merely represents Mr.
Bryan raised several powers. The same is true of the Populist as
compared to the Democratic platform. Mr. Bryan may affect to
believe that free silver does represent the ultimate goal, and that his
friends do not intend to go further in the direction of fiat money. Mr.
Watson’s friends, the middle-of-the-road Populists, are much more
fearless and much more logical. They are willing to accept silver as a
temporary makeshift, but they want a currency based on corn and
cotton next, and ultimately a currency based on the desires of the
people who issue it. The statesmanlike utterance of that great
financier, Mr. Bryan’s chief rival for the nomination and at present his
foremost supporter, Mr. Bland, to the effect that he would “wipe out
the national debt as with a sponge,” meets with their cordial approval
as far as it goes, but they object to the qualification before the word
“debt.” In wiping out debts they do not wish to halt merely at the
national debt. The Populists indorsed Bryan as the best they could
get; but they hated Sewall so that they took the extraordinary step of
nominating the Vice-President before the President so as to make
sure of a really acceptable man in the person of Watson.
With Mr. Bryan denunciation of the gold bug and the banker is
largely a mere form of intellectual entertainment; but with Mr. Watson
it represents an almost ferocious conviction. Someone has said that
Mr. Watson like Mr. Tillman, is an embodied retribution on the South
for having failed to educate the cracker, the poor white who gives
him his strength. It would ill beseem any dweller in cities of the
North, especially any dweller in the city of Tammany, to reproach the
South with having failed to educate anybody. But Mr. Watson is
certainly an awkward man for a community to develop. He is
infinitely more in earnest than is Mr. Bryan. Mr. Watson’s followers
belong to that school of southern Populists who honestly believe that
the respectable and commonplace people who own banks, railroads,
dry-goods stores, factories, and the like, are persons with many of
the mental and social attributes that unpleasantly distinguished
Heliogabalus, Nero, Caligula, and other worthies of later Rome. Not
only do they believe this, but they say it with appalling frankness.
They are very sincere as a rule, or at least the rank and file are.
They are also very suspicious. They distrust anything they cannot
understand; and as they understand but little this opens a very wide
field for distrust. They are apt to be emotionally religious. If not, they
are then at least atheists of an archaic type. Refinement and comfort
they are apt to consider quite as objectionable as immorality. That a
man should change his clothes in the evening, that he should dine at
any other hour than noon, impress these good people as being
symptoms of depravity instead of merely trivial. A taste for learning
and cultivated friends, and a tendency to bathe frequently, cause
them the deepest suspicion. A well-to-do man they regard with
jealous distrust, and if they cannot be well-to-do themselves, at least
they hope to make matters uncomfortable for those that are. They
possess many strong, rugged virtues, but they are quite impossible
politically, because they always confound the essentials and the non-
essentials, and though they often make war on vice, they rather
prefer making war upon prosperity and refinement.
Mr. Watson was in a sense born out of place when he was born in
Georgia, for in Georgia the regular Democracy, while it has accepted
the principles of the Populists, has made war on their personnel, and
in every way strives to press them down. Far better for Mr. Watson
would it have been could he have been born in the adjacent State of
South Carolina, where the Populists swallowed the Democrats with a
gulp. Senator Tillman, the great Populist or Democratic orator from
South Carolina, possesses an untrammelled tongue any middle-of-
the-road man would envy: and moreover Mr. Tillman’s brother has
been frequently elected to Congress upon the issue that he never
wore either an overcoat or an undershirt, an issue which any
Populist statesman finds readily comprehensible, and which he
would recognize at first glance as being strong before the people. It
needs a certain amount of mental subtlety to appreciate that it is for
one’s interest to support a man because he is honest and has broad
views about coast defenses and the navy, and other similar subjects;
but it does not need any mind at all to have one’s prejudices stirred
in favor of a statesman whose claim to the title rests upon his
indifference to the requirements of civilized dress.
Altogether Mr. Watson, with his sincerity, his frankness, his
extreme suspiciousness, his distrust of anything he cannot
understand, and the feeling he encourages against all the elegancies
and decencies of civilized life, is an interesting personage. He
represents the real thing, while Bryan after all is more or less a sham
and a compromise. Mr. Watson would, at a blow, destroy all banks
and bankers, with a cheerful, albeit vague, belief that thereby he was
in some abstruse way benefiting the people at large. And he would
do this with the simple sincerity and faith of an African savage who
tries to benefit his tribe by a sufficiency of human sacrifices. But Mr.
Bryan would be beset by ugly doubts when he came to put into effect
all the mischievous beliefs of his followers, and Mr. Sewall would
doubtless be frankly miserable if it ever became necessary for him to
take a lead in such matters. Mr. Watson really ought to be the first
man on the ticket, with Mr. Bryan second; for he is much the superior
in boldness, in thorough-going acceptance of his principles
according to their logical conclusions, and in sincerity of faith. It is
impossible not to regret that the Democrats and Populists should not
have put forward in the first place the man who genuinely represents
their ideas.
However, it is even doubtful whether Mr. Watson will receive the
support to which he is entitled as a vice-presidential candidate. In the
South the Populists have been so crushed under the heel of the
Democrats, and have bitten that heel with such eager venom, that
they dislike entering into a coalition with them; but in the south the
Democrats will generally control the election machinery. In the far
West, and generally in those States where the Populist wing of the
new alliance is ascendant, the Populists have no especial hatred of
the Democrats. They know that their principles are substantially
identical, and they think it best to support the man who seems to
represent the majority faction among the various factions that stand
behind Bryan.
As a consequence of this curious condition of affairs there are
several interesting possibilities open. The electoral college consists
of the men elected at the polls in the various States to record the
decrees of the majorities in those States, and it has grown to be an
axiom of politics that they must merely register the will of the men
who elected them. But it does seem possible that in the present
election some of the electors may return to the old principles of a

Biophysical Characterization of Proteins in Developing Biopharmaceuticals Second Edition Edition Damian J Houde Editor Full Chapter

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Biophysical characterization of proteins

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Critical to the development of any scientiﬁc developments and to the realiza-

(T)RPS (Tunable) resistive pulse sensing

cIEF Capillary isoelectric focusing

LMW Low molecular weight

TDA Taylor dispersion analysis

1.1 The basics of protein higher order structure (HOS)

Biophysical Characterization of Proteins in Developing

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.1.1 The levels of protein HOS

I. Proteins and biophysical characterization in the biopharmaceutical industry

I. Proteins and biophysical characterization in the biopharmaceutical industry

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.1.2 Stabilizing the HOS of proteins

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.1.3 Dynamics properties of a Protein’s HOS

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.1.4 Finer structural alteration of proteins

1.2 The search for how proteins attain their correct

I. Proteins and biophysical characterization in the biopharmaceutical industry

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.2.1 In vivo production of proteins: revisiting the protein folding problem

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.2.2 In vivo production of proteins: avoiding and eliminating folding errors

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.3 Surprises in the world of protein folding: intrinsically disordered or

1.4 Proteins and the biopharmaceutical industry: problems and

I. Proteins and biophysical characterization in the biopharmaceutical industry

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.4.1 Impact of PTMs on the HOS of protein biopharmaceuticals

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.4.2 Impact of changes in noncovalent interactions (secondary bonds) on the

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.4.3 A more detail discussion concerning protein biopharmaceutical

I. Proteins and biophysical characterization in the biopharmaceutical industry

1.4.4 The novelty of different classes of protein biopharmaceuticals that create

1.4.4.1 Example 1: Fc fusion proteins

I. Proteins and biophysical characterization in the biopharmaceutical industry

The Vice-President is an officer unique in his character and

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