Biophysical Characterization of Proteins in Developing Biopharmaceuticals 2Nd Edition Damian J Houde Editor Full Chapter

Biophysical Characterization of
Proteins in Developing
Biopharmaceuticals 2nd Edition
Damian J. 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.
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

an Fc fragment, it is hoped a similar effect of increased circulation time will be achieved. As

an example, fusing an Fc to the blood-clotting protein’s Factor VIII (FVIII-Fc) and Factor IX
(FIX-Fc) have been shown to reduce the clearance of these factors, while retaining the correct
biological activity [102e105]. This should enable patients suffering from Hemophilia A/B to
reduce the number of drug infusions. However, the fusion of two relatively large proteins
(each > 50 kDa) beckons the questions, “does the fusion of these two proteins cause any sig-
nificant changes to either protein that would lead to an alteration in their corresponding
HOS?” and, “would that fusion impact the functionality of either part of the molecule or
potentially lead to an adverse effect?”. While the answer to these questions will be protein-
dependent, for FVIII-Fc and FIX-Fc, the answer is no, as revealed by a battery of biophysical
studies [106e108].
1.4.4.2 Example 2: PEGylated proteins and antibody drug conjugates (ADCs)

The chemical coupling of a polyethylene glycol polymer (PEG) to a protein biopharmaceu-
tical yields a pegylated protein drug [94,95,109]. As observed with Fc fusion protein, pegy-
lated proteins show a significant reduction in the clearance of the administrated modified
protein drug from the body [109], significantly increasing their therapeutic value. However,
just as in the case of Fc fusion proteins the conjugation of the PEG molecule to a protein
beckons the same question concerning the impact of this modification on the HOS of the
modified protein (as discussed in the previous section) and for the same reasons. Neverthe-
less, to date there are currently more than a dozen pegylated biopharmaceuticals approved
and marketed [110] and more in development [111].
Similar situation exists for ADCs where a small generally toxic drug (called a payload) is
covalently bound to a mAb that uniquely bind to a specific cell (typically a cancerous cell
[96]). In this situation, the normally nonspecific toxic small molecule drug is turned into a
highly specific targeting drug by taking advantage of the high specificity of the mAb. The
mAb essentially guides (carries) the toxic pharmaceutical payload to its cellular targets
(e.g., cancer cell) where it’s internalized by the target cell and activated by its cleavage
from the ADC causing highly specific cell (e.g. cancer cell) killing. In constructing these
ADCs careful biophysical characterization studies are required to assess the impact of the
small pharmaceutical and its load (drug to antibody ratio, DAR) on the mAb [112] to insure
its overall HOS [113] (binding specificity) is not compromised.
1.4.4.3 Example 3: viruses, VLPs

The formation of very large (MDa) multisubunit protein complexes, such as a virus (e.g.,
used in gene therapy), VLP, nanoparticles (e.g., lipid nanoparticles, LNPs, liposomes, and
exosomes) which are used as drug delivery systems present unique challenges when trying
to biophysically characterize these complex biopharmaceuticals, which may be only partially
composed of protein material (or even in some cases may not contain any proteins, e.g., non-
protein biopharmaceuticals composed of specific nucleic acid molecules incased in LNPs or
Liposomes). A critical challenge in developing these very large biopharmaceuticals is in
assessing their homogeneity and overall structure. Due to their large size, the common work-
horse tool for acquiring this information, size-exclusion chromatograph (SEC, see Chapter 7),
may have limited utility because of limitations in its separation range (requiring SEC columns
with pore sizes that are often larger than what is commercially commonly found to be

References 21
available, i.e., >1000 Å). In this case, alternative analytical tools such as asymmetric flow field
flow fractionation (AF4, Chapters 10 and 15), analytical ultracentrifugation (AUC, Chapter 9),
and nanoparticle tracking analysis (NTA, Chapter 10) and flow cytometry (Chapter 10) can
be important biophysical tools capable of filling in this gap. Some of these tools, such as
AUC can also provide additional characterization information (chemical composition), which
is unique to these classes of very large complex biopharmaceuticals, e.g., the level of virus
drug particles that are filled, partially filled empty in terms of nucleic acid material [114].
1.5 Conclusion
In this chapter, the authors have provided, in broad strokes, brief discussions on the funda-
mental structural properties of proteins. Since a protein’s structure dictates its function, these
properties empower proteins with important functional roles for maintaining the cascade of
activities that characterize all living systems. However, these same structural properties also
create a heavy but required characterization workload for the biophysical scientist working in
the biopharmaceuticals industry. It is our hope that the reader is in a better position to
understand the unique challenges the biopharmaceutical industry encounters in striving to
bring these protein drugs to the market place. These challenges are significantly more daunt-
ing compared with those typically encountered in the pharmaceutical area, where the drug
product corresponds to small organic molecules with much simpler, rigid and homogenous
structure.
The developments that have occurred since the discovery of the structure of DNA [115], a
little over a half century ago that ushered in the molecular biology era, has culminated in the
last four decades with the successful commercial development of today’s growing biophar-
maceutical industry. Concurrent with this development has been the advancement of the bio-
analytical sciences, which has led to the development of better instruments and methods. Not
only are we able to better understand how these complex molecules work, but we are also
capable of characterizing them to better assure their safety and consistency of manufacturing.
In the area of biophysical characterization, significant innovative developments in newer
biophysical tools and techniques continue to occur along with the improvements in the older
and traditional biophysical tools and techniques. This situation makes our ability to charac-
terize the HOS of biopharmaceuticals on a routine level truly impressive. However, today
knowing what we do know versus knowing what we don’t know can be very sobering! It
seems, to the authors, that the more we discover the less we realize we know. Although still
lacking in our ability to characterize (biophysically) these fascinating protein drugs, the good
news is we are moving forward, learning to do a better job in participating in the overall task
of making more effective and safer drugs!
References
[1] Ramachandran GN, Ramakrishnan C, Sasisekharan V. Stereochemistry of polypeptide chain configurations.
J Mol Biol 1963;7:95e9.
[2] Creighton TE. The folded conformations of globular proteins. In: Proteins: structures and molecular properties.
New York (NY): W.H. Freeman and Co.; 1992.

[3] Creighton TE. The biophysical chemistry of nucleic acids & proteins. Helvetian Press; 2010.
[4] Kessel A, Ben-Tal N. Primary structure [Chapter 2]. In: Introduction to proteins: structure function and motion.
Boca Raton (FL): CRC Press; 2010.
[5] Kessel A, Ben-Tal N. Secondary structure. In: Introduction to proteins: structure function and motion. Boca
Raton (FL): CRC Press; 2010.
[6] Kyte J. Structure in protein chemistry. New York (NY): Garland Publishing, Inc.; 2006.
[7] Kessel A, Ben-Tal N. Tertiary structure. In: Introduction to proteins: structure function and motion. Boca Raton
(FL): CRC Press; 2010.
[8] Creighton TE. The physical and chemical basis of molecular biology. Helvetian Press; 2010.
[9] Creighton TE. The folded conformations of globular proteins. In: Proteins: structures and molecular properties.
New York (NY): W.H. Freeman and Co.; 2010.
[10] Houde DJ, Bou-Assaf GM, Berkowitz SA. Deciphering the biophysical effects of oxidizing sulfur-containing
amino acids in interferon-beta-1a using MS and HDX-MS. J Am Soc Mass Spectrom 2017;28(5):840e9.
[11] Bobst CE, Abzalimov RR, Houde D, Kloczewiak M, Mhatre R, Berkowitz SA, Kaltashov IA. Detection and
characterization of altered conformations of protein pharmaceuticals using complementary mass
spectrometry-based approaches. Anal Chem 2008;80(19):7473e81.
[12] Henzler-Wildman K, Kern D. Dynamic personalities of proteins. Nature 2007;450:964e72.
[13] Smock RG, Gierasch LM. Sending signals dynamically. Science 2009;324:198e203.
[14] Teilum K, Olsen JG, Kragelund BB. Functional aspects of protein flexibility. Cell Mol Life Sci 2009;66:2231e47.
[15] Tokuriki N, Tawfik DS. Protein dynamism and evolvability. Science 2009;324:203e7.
[16] Kamerzell TJ, Middaugh CR. The complex inter-relationships between protein flexibility and stability. J Pharm
Sci 2008;97:3494e517.
[17] Kamerzell TJ, Ramsey JD, Middaugh CR. Immunoglobulin dynamics, conformational fluctuations, and
nonlinear elasticity and their effects on stability. J Phys Chem 2008;112:3240e50.
[18] Liu H, May K. Disulfide bond structures of IgG molecules: structural variations, chemical modifications and
possible impacts to stability and biological function. mAbs 2012;4:17e23.
[19] Chen PL, Wang M, Ou WC, Lii CK, Chen LS, Chang D. Disulfide bonds stabilize JC virus capsid-like structure
by protecting calcium ions from chelation. FEBS Lett 2001;500:109e13.
[20] Toichi K, Yamanaka K, Furukawa Y. Disulfide scrambling describes the oligomer formation of superoxide
dismutase (SOD1) proteins in the familial form of amyotrophic lateral sclerosis. J Biol Chem
2013;288:4970e80.
[21] Trivedi MV, Laurence JS, Siahaan TJ. The role of thiols and disulfides on protein stability. Curr Protein Pept Sci
2009;10:614e25.
[22] Ho SC, Wang T, Song Z, Yang Y. IgG aggregation mechanism for CHO cell lines expressing excess heavy
chains. Mol Biotechnol 2015;57(7):625e34.
[23] Sakurai K, Nakahata R, Lee YH, Kardos J, Ikegami T, Goto Y. Effects of a reduced disulfide bond on aggrega-
tion properties of the human IgG1 CH3 domain. Biochim Biophys Acta 2015;854(10 Pt A).
[24] Nag M, Bera K, Basak S. Intermolecular disulfide bond formation promotes immunoglobulin aggregation:
investigation by fluorescence correlation spectroscopy. Proteins 2015;83(1):169e77.
[25] Moritz B, Stracke JO. Assessment of disulfide and hinge modifications in monoclonal antibodies. Electropho-
resis 2017;38(6):769e85.
[26] Morgan GJ, Usher GA, Kelly JW. Incomplete refolding of antibody light chains to non-native, protease-sensitive
conformations leads to aggregation: a mechanism of amyloidogenesis in patients. Biochemistry
2017;56(50):6597e614.
[27] Serebryany E, Woodard JC, Adkar BV, Shabab M, King JA, Shakhnovich EI. An internal disulfide locks a
misfolded aggregation-prone intermediate in cataract-linked mutants of human gD-crystallin. J Biol Chem
2016;291(36):19172e83.
[28] Kessel A, Ben-Tal N. Protein structural dynamics. Introduction to proteins: structure function and motion. Boca
Raton (FL): CRC Press; 2010.
[29] Bee JS, Randolph TW, Carpenter JF, Bishop SM, Dimitrova MN. Effects of surfaces and leachables on the sta-
bility of biopharmaceuticals. J Pharm Sci 2011;100(10):4158e70.
[30] Majumdar S, Ford BM, Mar KD, Sullivan VJ, Ulrich RG, D’Souza AJ. Evaluation of the effect of syringe surfaces
on protein formulations. J Pharm Sci 2011;100:2563e73.

References 23
[31] Sharma B. Immunogenicity of therapeutic proteins. Part 2: impact of container closures. Biotechnol Adv
2007;25:318e24.
[32] Sharma B. Immunogenicity of therapeutic proteins. Part 3: impact of manufacturing changes. Biotechnol
Adv 2007;25:325e31.
[33] Sharma B. Immunogenicity of therapeutic proteins. Part 1: impact of product handling. Biotechnol Adv
2007;25:310e7.
[34] Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, et al. Inherent toxicity of aggregates implies a
common mechanism for protein misfolding diseases. Nature 2002;416:507e11.
[35] Filipe V, Hawe A, Schellekens H, Jiskoot W. Aggregation and immunogenicity of therapeutic proteins. In: (ed-
itors: Wang W, Roberts CJ.) Aggregation of therapeutic proteins. John Wiley and Sons: Hoboken (NY), pp.
400e33.
[36] Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J 2006;8:E501e7.
[37] Walsh CT. Posttranslational modification of proteins: expanding nature’s inventory. Englewood , CO: Roberts
and Co.; 2006. 490pp.
[38] Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, et al. Essentials of glycobiology. Cold
Springs Harbor (NY): Cold Springs Harbor Laboratory Press; 2009.
[39] Kessel A, Ben-Tal N. Protein structure. Introduction to proteins: structure function and motion, CRC Press,
Boca Raton (FL).
[40] Creighton TE. Biosynthesis of proteins. Proteins: structures and molecular properties, W.H. Freeman and Co.,
New York (NY).
[41] Walsh CT, Garneau-Tsodikova S, Gatto Jr GJ. Protein posttranslational modifications: the chemistry of prote-
ome diversifications. Angew Chem Int Ed 2005;44:7342e72.
[42] Farley AR, Link AJ. Identification and quantification of protein posttranslational modifications. Methods
Enzymol 2009;463:725e63.
[43] Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol
2006;24:1241e52.
[44] Anfinsen CB. Principles that govern the folding of protein chains. Science 1973;181:223e30.
[45] Dill KA, MacCallum JL. The protein-folding problem, 50 years on. Science 2012;338:1042e6.
[46] Levinthal C. How to fold graciously. Mossbauer Spectrosc Biol Syst 1969;67:22e6 [Proceedings].
[47] Bryngelson JD, Onuchic JN, Socci ND, Wolynes PG. Funnels, pathways, and the energy landscape of protein
folding: a synthesis. Proteins 1995;21:167e95.
[48] Dill KA, Chan HS. From Levinthal to pathways to funnels. Nat Struct Biol 1997;4:10e9.
[49] Dill KA, Ozkan SB, Shell MS, Weikl TR. The protein folding problem. Annu Rev Biophys 2008;37:289e316.
[50] Onuchic JN, Wolynes PG. Theory of protein folding. Curr Opin Struct Biol 2004;14:70e5.
[51] Onuchic JN, Wolynes PG, Luthey-Schulten Z, Socci ND. Toward an outline of the topography of a realistic
protein-folding funnel. Proc Natl Acad Sci USA 1995;92:3626e30.
[52] Panchenko AR, Luthey-Schulten Z, Wolynes PG. Foldons, protein structural modules, and exons. Proc Natl
Acad Sci USA 1996;93:2008e13.
[53] Englander SW, Mayne L, Krishna MM. Protein folding and misfolding: mechanism and principles. Q Rev
Biophys 2007;40:287e326.
[54] Fedorov AN, Baldwin TO. Protein folding and assembly in a cell-free expression system. Methods Enzymol
1998;290:1e17.
[55] Netzer WJ, Hartl FU. Recombination of protein domains facilitated by co-translational folding in eukaryotes.
Nature 1997;388:343e9.
[56] Cabrita LD, Dobson CM, Christodoulou J. Protein folding on the ribosome. Curr Opin Struct Biol
2010;20:33e45.
[57] Fedorov AN, Baldwin TO. Cotranslational protein folding. J Biol Chem 1997;272:32715e8.
[58] Beissinger M, Buchner J. How chaperones fold proteins. Biol Chem 1998;379:245e59.
[59] Gottesman S, Wickner S, Maurizi MR. Protein quality control: triage by chaperones and proteases. Genes Dev
1997;11:815e23.
[60] Lee S, Tsai FT. Molecular chaperones in protein quality control. J Biochem Mol Biol 2005;38:259e65.
[61] Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev 2013;13:630e42.

[62] Quan S, Bardwell JC. Chaperone discovery. Bioessays 2012;34:973e81.

[63] Sorokin AV, Kim ER, Ovchinnikov LP. Proteasome system of protein degradation and processing. Biochem-
istry (Mosc) 2009;74:1411e42.
[64] Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, et al. Intrinsically disordered protein. J Mol
Graph Model 2001;19:26e59.
[65] Dunker AK, Babu MM, Barbar E, Blackledge M, Bondos SE, Dosztanyi Z, et al. What’s in a name? Why these
proteins are intrinsically disordered. Intrinsically Disord Proteins 2013;1:e24157.
[66] Huang Y-M, Hu W, Rustandi E, Chang K, Yusuf-Makagiansar H, Ryll T. Maximizing productivity of CHO cell-
based fed-batch culture using chemically defined media conditions and typical manufacturing equipment. Bio-
technol Prog 2010;26:1400e10.
[67] Britt KA, Schwartz DK, Wurth C, Mahler HC, Carpenter JF, Randolph TW. Excipient effects on humanized
monoclonal antibody interactions with silicone oil emulsions. J Pharm Sci 2012;101:4419e32.
[68] DiMasi JA, Grabowski HG, Hansen RW. Innovation in the pharmaceutical industry new estimates of R&D.
J Health Econ 2016;47:20e32.
[69] Smith LM, Kelleher NL. Proteoform: a single term describing protein complexity. Nat Methods
2013;10(3):186e7.
[70] Ha E, Wang W, Wang YJ. Peroxide formation in polysorbate 80 and protein stability. J Pharm Sci
2002;91:2252e64.
[71] Jiang Y, Nashed-Samuel Y, Li C, Liu W, Pollastrini J, Mallard D, et al. Tungsten-induced protein aggregation:
solution behavior. J Pharm Sci 2009;98:4695e710.
[72] Kerwin BA, Remmele Jr RL. Protect from light: photodegradation and protein biologics. J Pharm Sci
2007;96:1468e79.
[73] Patel J, Kothari R, Tunga R, Ritter NM, Tunga BS. Stability considerations for biopharmaceuticals, Part I.
Bioprocess Int 2011:20e31.
[74] Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm
1999;185:129e88.
[75] Bossio RE, Marshall AG. Baseline resolution of isobaric phosphorylated and sulfated peptides and nucleotides
by electrospray ionization FTICR ms: another step toward mass spectrometry-based proteomics. Anal Chem
2002;74:1674e9.
[76] Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in pep-
tides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem 1987;262:785e94.
[77] Hambly DM, Banks DD, Scavezze JL, Siska CC, Gadgil HS. Detection and quantitation of IgG 1 hinge aspartate
isomerization: a rapid degradation in stressed stability studies. Anal Chem 2009;81:7454e9.
[78] Czajkowsky DM, Hu J, Shao Z, Pleass RJ. Fc-fusion proteins: new developments and future perspectives.
EMBO Mol Med 2012;4(10):1015e28.
[79] Beck A, Reichert JM. Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. mAbs
2011;3:415e6.
[80] Philo JS, Arakawa T. Mechanisms of protein aggregation. Curr Pharmaceut Biotechnol 2009;10:348e51.
[81] Chi EY, Krishnan S, Kendrick BS, Chang BS, Carpenter JF, Randolph TW. Roles of conformational stability and
colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor. Protein Sci
2003;12:903e13.
[82] Mahler HC, Fischer S, Randolph TW, Carpenter JF. Protein aggregation and particle formation effects of formu-
lation, interfaces, and drug product manufacturing operations. In: Wang W, Roberts CJ, editors. Aggregation of
therapeutic proteins. John Wiley & Sons, Inc.; 2010. p. 301e5.
[83] Ellis RJ, Minton AP. Protein aggregation in crowed environments. Biol Chem 2006;387:485e97.
[84] Minton AP. Influence of macromolecular crowding upon the stability and state of association of proteins: pre-
dictions and observations. J Pharm Sci 2005;94:1668e75.
[85] Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm
Sci 2004;93:1390e402.
[86] Demeule B, Shire SJ, Liu J. A therapeutic antibody and its antigen form different complexes in serum than in
phosphate-buffered saline: a study by analytical ultracentrifugation. Anal Biochem 2009;388:279e87.
[87] Wright RT, Hayes DB, Stafford WF, Sherwood PJ, Correia JJ. Characterization of therapeutic antibodies in the
presence of human serum proteins by AU-FDS analytical ultracentrifugation. Anal Biochem 2018;550:72e83.

References 25
[88] Mathaes R, Koulov A, Joerg S, Mahler HC. Subcutaneous injection volume of biopharmaceuticalsdpushing the
boundaries. J Pharm Sci 2016;105(8):2255e9.
[89] Chaudhri A, Zarraga IE, Yadav S, Patapoff TW, Shire SJ, Voth GA. The role of amino acid sequence in the self-
association of therapeutic monoclonal antibodies: insights from coarse-grained modeling. J Phys Chem
2013;117:1269e79.
[90] Kayser V, Chennamsetty N, Voynov V, Helk B, Forrer K, Trout BL. Evaluation of a non-Arrhenius model for
therapeutic monoclonal antibody aggregation. J Pharm Sci 2011;100:2526e42.
[91] Li S, Xing D, Li J. Dynamic light scattering application to study protein interactions in electrolyte solutions.
J Biol Phys 2004;30:313e24.
[92] Scherer TM, Liu J, Shire SJ, Minton AP. Intermolecular interactions of IgG1 monoclonal antibodies at high
concentrations characterized by light scattering. J Phys Chem 2010;114:12948e57.
[93] Yadav S, Scherer TM, Shire SJ, Kalonia DS. Use of dynamic light scattering to determine second virial coeffi-
cient in a semidilute concentration regime. Anal Biochem 2011;411:292e6.
[94] Bailon P, Won CY. PEG-modified biopharmaceuticals. Expert Opin Drug Deliv 2009;6:1e16.
[95] Jevsevar S, Kunstelj M, Porekar VG. PEGylation of therapeutic proteins. Biotechnol J 2010;5:113e28.
[96] Lambert JM, Berkenblit A. Antibody-drug conjugates for cancer treatment. Annu Rev Med 2018;69:191e207.
[97] Podust VN, Balan S, Sim BC, Coyle MP, Ernst U, Peters RT, Schellenberger V. Extension of in vivo half-life of
biologically active molecules by XTEN protein polymers. J Control Release 2016;240:52e66.
[98] Ma Y, Nolte RJM, Cornelissen JJLM. Virus-based nanocarriers for drug delivery. Adv Drug Deliv Rev
2012;64:811e25.
[99] Unverdorben F, Richter F, Hutt M, Seifert O, Malinge P, Fischer N, Kontermann RE. Pharmacokinetic proper-
ties of IgG and various Fc fusion proteins in mice. mAbs 2016;8(1):120e8.
[100] Roopenian DC, Akilesh S. FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol 2007;7:715e25.
[101] Roopenian DC, Christianson GJ, Sproule TJ, Brown AC, Akilesh S, Jung N, et al. The MHC class I-like IgG
receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs. J Immunol
2003;170:3528e33.
[102] Peters RT, Low SC, Kamphaus GD, Dumont JA, Amari JV, Lu Q, et al. Prolonged activity of factor IX as a
monomeric Fc fusion protein. Blood 2010;115:2057e64.
[103] Shapiro AD, Ragni MV, Valentino LA, Key NS, Josephson NC, Powell JS, et al. Recombinant factor IX-Fc fusion
protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients.
Blood 2012;119:666e72.
[104] Dumont JA, Liu T, Low SC, Zhang X, Kamphaus G, Sakorafas P, et al. Prolonged activity of a recombinant
factor VIII-Fc fusion protein in hemophilia A mice and dogs. Blood 2012;119:3024e30.
[105] Powell JS, Josephson NC, Quon D, Ragni MV, Cheng G, Li E, et al. Safety and prolonged activity of recombi-
nant factor VIII Fc fusion protein in hemophilia A patients. Blood 2012;119:3031e7.
[106] Houde D, Berkowitz SA. Conformational comparability of factor IX-Fc fusion protein, factor IX, and purified Fc
fragment in the absence and presence of calcium. J Pharm Sci 2012;101:1688e700.
[107] Li H, Bai S, Wei JY, Berkowitz SA, Brader ML. Calcium binding to a factor ix Fc fusion protein and effects on
higher-order structure. J Pharm Sci 2011;100:4597e606.
[108] Kulman J, et al. Structural comparability between recombinant FVIII-Fc and its isolated FVIII and Fc
constituents, Abstract 1135 presented at the 54th Annual meeting and exposition in Altlanta Ga 8e11 December
2012.
[109] Harris JM, Martin NE, Modi M. Pegylation: a novel process for modifying pharmacokinetics. Clin Pharmaco-
kinet 2001;40:539e51.
[110] Turecek PL, Bossard MJ, Schoetens F, Ivens IA. PEGylation of biopharmaceuticals: a review of chemistry and
nonclinical safety information of approved drugs. J Pharm Sci 2016;105(2):460e75.
[111] Ivens IA, Achanzar W, Baumann A, Brändli-Baiocco A, Cavagnaro J, et al. PEGylated biopharmaceuticals:
current experience and considerations for nonclinical development. Toxicol Pathol 2015;43(7):959e83.
[112] Adem YT, Schwarz KA, Duenas E, Patapoff TW, Galush WJ, Esue O. Auristatin antibody drug conjugate
physical instability and the role of drug payload. Bioconjug Chem 2014;25(4):656e64.
[113] Pan LY, Salas-Solano O, Valliere-Douglass JF. Conformation and dynamics of interchain cysteine-linked
antibody-drug conjugates as revealed by hydrogen/deuterium exchange mass spectrometry. Anal Chem
2014;86(5):2657e64.

[114] Berkowitz SA, Philo JS. Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery
system) using analytical ultracentrifugation. Anal Biochem 2007;362:16e37.
[115] Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature
1953;171:737e8.
[116] Berkowitz SA. Analytical characterization: structural assessment of biosimilarity. In: Endrenyi L, Declerck P,
Chow S-C, editors. Biosimilar drug product development, vol. 216. Bca Raton, FL: CRC Press; 2015. p. 15e82.
Further reading
For a comprehensive review on many of the topics covered in this chapter, the authors highly recommend the following
useful books:
[1] Creighton TE. Proteins: Structures and Molecular Properties. 2nd ed. W.H. Freeman and Co., NY.
[2] Creighton TE. The Biophysical Chemistry of Nucleic Acids & Proteins, Helvetian Press.
[3] Cantor CR, Schimmel PR. Biophysical Chemistry Part I: The Conformation of Biological Macromolecules, W.H.
Freeman & Company, New York.
[4] Dickerson RE, Geis I. The Structure and Action of Proteins, Harper & Row, New York.
[5] Kaltashov IA, Eyles SJ. Mass Spectrometry in Biophysics, Wiley-Interscience, Hoboken (NJ).
[6] Kessel A, Ben-Tal N. Introduction to Proteins: Structure, Function and Motion, CRC Press, Boca Raton (FL).
[7] Kyte J. Structure in Protein Chemistry, Garland Publishing, Inc., NY.

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a point which His Majesty's Government can afford to concede.
I think it would have a deplorable effect in Cape Colony and
Natal to obtain peace by such a concession." Mr. Chamberlain
agreed with the High Commissioner, writing in reply: "His
Majesty's Government feel that they cannot promise to ask for
complete amnesty to Cape and Natal rebels who are in totally
different position to burghers without injustice to those who
have remained loyal under great provocation, and they are
prepared substantially to adopt your words, but you must
consider whether your last line is strictly applicable to
Natal." Mr. Chamberlain made numerous other criticisms of Lord
Kitchener's suggested letter, and amended it in many
particulars, the most important of which related to the form
of government under which the late republics would be placed.
Lord Kitchener would have said: "Military law will cease and
be at once replaced by civil administration, which will at
first consist of a Governor and a nominated Executive with or
without an advisory elected Assembly, but it is the desire of
His Majesty's Government, as soon as circumstances permit, to
establish representative Government in the Transvaal and
Orange River Colony." His political superior instructed him to
change the statement as follows: "For 'military law will
cease' say 'military administration will cease.' It is
possible that there may be disturbed districts for some time
after terms have been accepted, and Governor of Colonies
cannot abandon right of proclaiming martial law where
necessary. In the same sentence omit the words 'at the same
time' and 'at once' and substitute at the beginning the words
'at the earliest practicable date.' For 'consist of a
Governor' down to 'Assembly' read 'consist of a Governor and
an Executive Council composed of the principal officials with
a Legislative Council consisting of a certain number of
official members to whom a nominated unofficial element will
from the first be added.' In place of the words 'to establish
representative government' substitute 'to introduce a
representative element, and ultimately to concede to the new
Colonies the privilege of self-government.' It is desirable at
this stage to be quite precise in order to avoid any charge of
breach of faith afterwards."
Out of the instructions he received, Lord Kitchener finally

framed the following letter to Commandant Botha, sent to him
on the 7th of March: "With reference to our conversation at
Middelburg on 28th February, I have the honour to inform you
that in the event of a general and complete cessation of
hostilities and the surrender of all rifles, ammunition,
cannon, and other munitions of war in the hands of the
burghers or in Government depots or elsewhere, His Majesty's
Government is prepared to adopt the following measures:
"His Majesty's Government will at once grant an amnesty in the

Transvaal and Orange River Colonies for all bona fide acts of
war committed during the recent hostilities. British subjects
belonging to Natal and Cape Colony, while they will not be
compelled to return to those Colonies, will, if they do so, be
liable to be dealt with by the law of those Colonies specially
passed to meet the circumstances arising out of the present
war. As you are doubtless aware, the special law in the Cape
Colony has greatly mitigated the ordinary penalties for high
treason in the present cases.
"All prisoners of war now in St. Helena, Ceylon, or elsewhere

will, on the completion of the surrender, be brought back to
their country as quickly as arrangements can be made for their
transport.
"At the earliest practicable date military administration will

cease and will be replaced by civil administration in the form
of Crown Colony Government. There will therefore be, in the
first instance, in each of the new Colonies a Governor and an
Executive Council, consisting of a certain number of official
members, to whom a nominated unofficial element will be added.
But it is the desire of His Majesty's Government, as soon as
circumstances permit, to introduce a representative element
and ultimately to concede to the new Colonies the privilege of
self-government. Moreover, on the cessation of hostilities a High
Court will be established in each of the new Colonies to
administer the law of the land, and this Court will be
independent of the Executive.
"Church property, public trusts, and orphans funds will be

respected.
"Both the English and Dutch languages will be used and taught
in public schools where parents of the children desire it, and
allowed in Courts of Law.
"As regards the debts of the late Republican Governments, His

Majesty's Government cannot undertake any liability. It is,
however, prepared, as an act of grace, to set aside a sum not
exceeding £1,000,000 to repay inhabitants of the Transvaal and
Orange River Colonies for goods requisitioned from them by the
late Republican Governments, or, subsequent to annexation, by
Commandants in the field being in a position to enforce such
requisitions. But such claims will have to be established to
the satisfaction of a Judge or Judicial Commission appointed
by the Government to investigate and assess them, and if
exceeding in the aggregate £1,000,000, they will be liable to
reduction pro rata.
"I also beg to inform your Honour that the new Government will
take into immediate consideration the possibility of assisting
by loan the occupants of farms who will take the oath of
allegiance to repair any injury sustained by destruction of
buildings or loss of stock during the war, and that no special
war tax will be imposed on farmers to defray the expense of
the war.
"When burghers require the protection of fire-arms such will

be allowed to them by licence and on due registration,
provided they take the oath of allegiance. Licences also will
be issued for sporting rifles, guns, &c., but military
firearms will only be allowed for means of protection.
{513}
"As regards the extension of the franchise to Kaffirs in the

Transvaal and Orange River Colony, it is not the intention of
His Majesty's Government to give such franchise before
representative government is granted to these Colonies, and if
then given it will be so limited as to secure the just
predominance of the white races. The legal position of
coloured persons will, however, be similar to that which they
hold in Cape Colony.
"In conclusion, I must inform your Honour that if the terms

now offered are not accepted after a reasonable delay for
consideration they must be regarded as cancelled."
On the 16th of March the following reply came from the Boer
Commandant: "I have the honour to acknowledge receipt of your
Excellency's letter stating what steps your Excellency's
Government is prepared to take in the event of a general and
total cessation of hostilities. I have advised my Government
of your Excellency's said letter; but, after the mutual
exchange of views at our interview at Middelburg on 28th
February last, it will certainly not surprise your Excellency
to know that I do not feel disposed to recommend that the
terms of the said letter shall have the earnest consideration
of my Government. I may add also that my Government and my
chief officers here entirely agree to my views." This ended
the negotiations.
A discussion of the negotiations in Parliament occurred on the

28th of March, when Mr. Bryce (Liberal) said "they were agreed
that the Government took an onward step when they allowed the
peace negotiations to be entered into, and it was important to
observe that, not only Lord Kitchener, but Sir Alfred Milner
was persuaded that General Botha meant business. It was
possible there were causes at work with which the House were
not acquainted which caused the negotiations to be broken off.
General Botha wrote to Lord Kitchener:—'You will not be
surprised to hear that my answer is in the negative.' One of
two things must have happened—either Lord Kitchener heard from
General Botha something that the House had not heard of, or
else General Botha was so much struck by the difference
between the terms which Lord Kitchener had discussed and the
terms contained in the letter that he conceived a distrust of
us altogether and believed that the Government would not
accept what Lord Kitchener had offered. He thought the
Government were right in asking that the oath of allegiance
should be taken, that they were entitled to insist upon the
provision that all hostilities must cease, and that they could
not pledge themselves as to the precise time when they would
bring back the prisoners. But there were three points on which
there were substantial differences between the terms Lord
Kitchener appeared to have offered and the terms in the final
letter. The first is the question of amnesty for the Cape
rebels. Lord Kitchener and General Botha appeared to have come
to an agreement on that subject. General Botha did not object
to the disfranchisement of the Cape rebels, and Lord Kitchener
did not appear to have conveyed any suggestion whatever of
anything except disfranchisement. He could conceive nothing
more likely to turn back the pacific desires of the Boers than
the fact that they found that, instead of the Cape rebels
having nothing but disfranchisement to fear, they were to be
held subject to the Cape laws as to treason. He was not
arguing whether that was right or wrong. The question was what
the Boers would think, and he put it to the House that it was
the most natural thing that they should be struck by the
contrast between the terms which Lord Kitchener appeared to
offer and the terms which were offered when the final letter
came, and that that was just the point upon which brave men,
feeling for their comrades, would be inclined to stand out.
They would be told that they would displease the loyalists at
the Cape if they did not exact all the penalties for treason.
He hoped they would never in that House consider it any part
of their business to satisfy the vindictive feeling of the
colonists at the Cape."
As to the difference between the terms of future government

for the inhabitants of the late republics proposed by Lord
Kitchener and those laid down by the Colonial Secretary, Mr.
Bryce said: "He should like to have known what the proposals
were that General Botha made with regard to a modified
independence, for he thought it was quite possible that it
might turn out in the long run that some kind of what was
called modified independence, protection, would be a great
deal easier for this country to work than a system of Crown
colony government. He thought the contrast between the
elective assembly which Lord Kitchener offered and the purely
arbitrary and despotic system which the final letter conveyed
must at once have struck the Boers as indicating the
difference between the views which the military man on the
spot entertained and the proposal which they might expect from
the Government. Of course there were objections to the
immediate grant of self-government. So also there were
objections to any course, and that course should be chosen
which was open to the fewest objections. But the proposal of
Crown colony government was, of all courses, the worst that
could be suggested. It had been suggested that members of the
Liberal party had asked for full-grown representative and
responsible government, but they never had suggested that.
What they had objected to was Crown colony government. They
admitted that when the war ended there must be an intermediate
period of administration, military or civil, but there was all
the difference in the world between an admittedly provisional
administration understood to be provisional and the creation
of the whole apparatus of Crown colony government. The Boer
population had an aversion to Crown colony administration,
associated in their minds with the days of Sir Owen Lanyon,
and an arbitrary form of government it was known to be. Of
course it was arbitrary; honourable members who questioned
that could not know what Crown colony administration was. The
existence of a nominated council did not prevent it being
arbitrary inasmuch as the members were obliged to vote as they
were directed by the Governor. He could not help thinking that
Lord Kitchener might, if he were asked to do so, throw some
light on a remarkable expression in the letter from General
Botha in which he said, after the mutual interchange of views
at their meeting, Lord Kitchener would not be surprised to
learn that he was not disposed to recommend the terms
proposed."
{514}
The radical Mr. Labouchere was sharper in his criticism: "He

held that it was nonsense to call the terms offered to the
Boers liberal and lenient; they were neither. We had burnt
their farms and desolated their country, and then we offered
them a small gift of money to put them back on their farms
while we took away their independence and their flag. He
honoured the men who resisted, no matter at what cost, when
the question was the independence of their native land. How
right General Botha was in distrusting the alterations made by
the Secretary for the Colonies in the matter of the gift was
shown by the right honourable gentleman himself, when he said
that, whereas the gift was to be limited to a certain sum, the
loyalists were to be paid first. In that case what would
remain to the burghers of the two colonies? The position of
the Boers in the Empire under the terms of the Colonial
Secretary would be little better than that of Kaffirs. As far
as ultimate self-government was concerned, they were to put
their faith in the Colonial Secretary. If he might offer them
a word of advice it would be—Put no faith in the Colonial
Secretary; get it in black and white. We had lost a great
opportunity of ending the war and settling South Africa. Peace
won by the sword would create a dependency in which racial
feuds would go on and the minority would be maintained over
the majority by a huge British garrison. The Dutch majority
was certain to increase every decade. The Transvaal farmers
lived in a poor, rude manner which English people would not
accept. …
"He did not particularly admire the Boers. To his mind they
had too much of the conservative element in them; but, judging
between the Afrikanders and the English who went to South
Africa, whilst fully recognizing that among the latter there
were many respectable men, he thought, taking them
collectively, the Boers were the better men. If we wanted to
maintain our rule in South Africa the Boers were the safest
men with whom to be on good terms. What were the Boers ready
to do? As he read the correspondence, they were ready to enter
the area of the British Empire, but only upon terms. Surely
our problem was to find terms honourable to us and to them,
which would lead to South Africa becoming one of those great
commonwealths connected with the Empire such as existed in
Australia and Canada. He suggested that, in the first place,
we should offer a full and absolute amnesty. He urged that the
Orange State and the Transvaal should as soon as possible be
made self-governing colonies. The Orange State was regarded by
every Englishman who had written about it as a model State. As
to the Transvaal, he admitted there was a difficulty, but he
would suggest that the main area of the country should be
separated from the Rand. The Rand might be administered by a
governor, a military governor if they liked, while in the rest
of the country the Dutch would have a majority. If this course
were adopted, instead of our giving some sort of pecuniary aid
to the Transvaalers, they might be paid a reasonable rent for
the Rand district, of which they would be deprived. … They on
that side of the House would be perfectly ready to agree to
the establishment of a provisional government, military or
civil—he should himself prefer Lord Kitchener to Sir Alfred
Milner—to carry on the country while they were arranging for
the colony to be self-governing. They were accustomed to be
told that Sir Alfred Milner was a sort of divine pro-consul.
He believed Sir Alfred Milner to be a most honourable man, and
very intelligent in many walks of life; but the truth was that
he began life as an Oxford don and then became an official in
the Treasury, facts which militated against his success in
practical politics. He believed that a man like Lord Dufferin
would do more for the cause of peace in South Africa than all
our soldiers."
SOUTH AFRICA: The Field of War: A. D. 1901 (February-April).

The High Commissioner, Sir Alfred Milner, on the situation
and prospects.
Leave of absence obtained by Sir Alfred.
A British Blue Book, made public in London on the 18th of

April, contains an interesting despatch from Sir Alfred
Milner, frankly reviewing the general situation in South
Africa, as it appeared to him on the 6th of February, when he
wrote, from Cape Town, and giving his forecast of future
prospects. The following are the more important passages of
the communication:
"A long time has elapsed since I have attempted to send to you
any general review of South African affairs. The reason is
twofold. In the first place, I am occupied every day that
passes from morning till night by business, all of which is
urgent, and the amount and variety of which you are doubtless
able to judge from the communications on a great variety of
subjects, which are constantly passing between us. In the next
place, I have always hoped that some definite point would be
reached at which it might be possible to sum up that chapter
of our history which contained the war, and to forecast the
work of administrative reconstruction which must succeed it.
But I am reluctantly forced to the conclusion that there will
be no such dividing line. I have not the slightest doubt of
the ultimate result, but I foresee that the work will be
slower, more difficult, more harassing, and more expensive
than was at one time anticipated. At any rate, it is idle to
wait much longer in the hope of being able to describe a clear
and clean-cut situation. Despite the many other calls upon my
time, and despite the confused character of the present
position, I think it better to attempt to describe, however
roughly and inadequately, the state of things as it exists
to-day.
"It is no use denying that the last half-year has been one of
retrogression. Seven months ago this Colony was perfectly
quiet, at least as far as the Orange River. The southern half
of the Orange River Colony was rapidly settling down, and even
a considerable portion of the Transvaal, notably the
south-western districts, seemed to have definitely accepted
British authority, and to rejoice at the opportunity of a
return to orderly government, and the pursuits of peace.
To-day the scene is completely altered. It would be
superfluous to dwell on the increased losses to the country
caused by the prolongation of the struggle, and by the form
which it has recently assumed. The fact that the enemy are now
broken up into a great number of small forces, raiding in
every direction, and that our troops are similarly broken up
in pursuit of them, makes the area of actual fighting, and
consequently of destruction, much wider than it would be in
the case of a conflict between equal numbers operating in
large masses.
{515}
Moreover, the fight is now mainly over supplies. The Boers
live entirely on the country through which they pass, not only
taking all the food they can lay hands upon on the farms,
grain, forage, horses, cattle, &c., but looting the small
village stores for clothes, boots, coffee, sugar, &c., of all
which they are in great need. Our forces, on their side, are
compelled to denude the country of everything moveable, in
order to frustrate these tactics of the enemy. No doubt a
considerable amount of the stock taken by us is not wholly
lost, but simply removed to the refugee camps, which are now
being established at many points along the railway lines. But
even under these circumstances, the loss is great, through
animals dying on the route, or failing to find sufficient
grass to live upon when collected in large numbers at the
camps. Indeed, the loss of crops and stock is a far more
serious matter than the destruction of farm buildings, of
which so much has been heard. I say this not at all as an
advocate of such destruction. I am glad to think that the
measure is now seldom if ever resorted to. At the same time,
the destruction of even a considerable number of farms, having
regard to the very rough and inexpensive character of the
majority of these structures in the Orange River Colony and
Transvaal, is a comparatively small item in the total damage
caused by the war to the agricultural community.
"To the losses incidental to the actual course of the

campaign, there has recently been added destruction of a
wholly wanton and malicious character. I refer to the injury
done to the head-gear, stamps, and other apparatus of some of
the outlying mines by Boer raiders, whose sole object was
injury. For this destruction there is, of course, no possible
excuse. … Fortunately the damage done to the mines has not
been large, relatively to the vast total amount of the fixed
capital sunk in them. The mining area is excessively difficult
to guard against purely predatory attacks having no military
purpose, because it is, so to speak, 'all length and no
breadth'—one long thin line, stretching across the country
from east to west for many miles. Still, garrisoned as
Johannesburg now is, it is only possible successfully to
attack a few points in it. Of the raids hitherto made, and
they have been fairly numerous, only one has resulted in any
serious damage. In that instance the injury done to the single
mine attacked amounted to £200,000, and it is estimated that
the mine is put out of working for two years. This mine is
only one out of a hundred, and is not by any means one of the
most important. These facts may afford some indication of the
ruin which might have been inflicted, not only on the
Transvaal and all South Africa, but on many European
interests, if that general destruction of mine works which was
contemplated just before our occupation of Johannesburg had
been carried out. However serious in some respects may have
been the military consequences of our rapid advance to
Johannesburg, South Africa owes more than is commonly
recognized to that brilliant dash forward, by which the vast
mining apparatus, the foundation of all her wealth, was saved
from the ruin threatening it.
"The events of the last six or seven months will involve a

greater amount of repair and a longer period of recuperation,
especially for agriculture, than anybody could have
anticipated when the war commenced. Yet, for all that, having
regard to the fact that both the Rand and Kimberley are
virtually undamaged, and that the main engines of prosperity,
when once set going again, will not take very long to get into
working order, the economic consequences of the war, though
grave, do not appear by any means appalling. The country
population will need a good deal of help, first to preserve it
from starvation, and then, probably, to supply it with a
certain amount of capital to make a fresh start. And the great
industry of the country will need some little time before it
is able to render any assistance. But, in a young country with
great recuperative powers, it will not take many years before
the economic ravages of the war are effaced.
"What is more serious to my mind than the mere material

destruction of the last six months is the moral effect of the
recrudescence of the war. I am thinking especially of the
Orange River Colony, and of that portion of the Transvaal
which fell so easily into our hands after the relief of
Mafeking, that is to say, the country lying between
Johannesburg and Pretoria, and the border of Bechuanaland.
Throughout this large area the feeling in the middle of last
year was undoubtedly pacific. The inhabitants were sick of the
war. They were greatly astonished, after all that had been
dinned into them, by the fair and generous treatment they
received on our first occupation, and it would have taken very
little to make them acquiesce readily in the new regime. At that
time too, the feeling in the Colony was better than I have
ever known it. The rebellious element had blown off steam in
an abortive insurrection, and was glad to settle down again.
If it had been possible for us to screen those portions of the
conquered territory, which were fast returning to peaceful
pursuits, from the incursions of the enemy still in the field,
a great deal of what is now most deplorable in the condition of
South Africa would never have been experienced. The vast
extent of the country, the necessity of concentrating our
forces for the long advance, first to Pretoria and then to
Komati Poort, resulted in the country already occupied being
left open to raids, constantly growing in audacity, and fed by
small successes, on the part of a few bold and skilful
guerrilla leaders who had nailed their colours to the mast.
The reappearance of these disturbers of the peace, first in
the south-east of the Orange River Colony, then in the
south-west of the Transvaal, and finally in every portion of
the conquered territory, placed those of the inhabitants who
wanted to settle down in a position of great difficulty.
Instead of being made prisoners of war, they had been allowed
to remain on their farms on taking the oath of neutrality, and
many of them were really anxious to keep it. But they had not the
strength of mind, nor, from want of education, a sufficient
appreciation of the sacredness of the obligation which they
had undertaken, to resist the pressure of their old companions
in arms when these reappeared among them appearing to their
patriotism and to their fears. …
{516}
"As the guerrilla warfare swept back over the whole of the
western Transvaal, and practically the whole of the Orange
River Colony, its effect upon the Cape Colony also became very
marked. There was a time, about the middle of last year, when
the bulk of the Dutch population in the Cape Colony, even
those who had been most bitter against us at the outset,
seemed disposed to accept the 'fait accompli,' and were
prepared to acquiesce in the union of all South Africa under
the British flag. Some of them even began to see certain
advantages in such a consummation. The irreconcilable line
taken in the Cape Parliament, during its recent Session from
July to October, was a desperate effort to counteract this
tendency. But I doubt whether it would have succeeded to the
moderate extent to which it has, had it not been for the
recrudescence of the war on the borders of the Colony, and the
embittered character which it assumed. Every act of harshness,
however necessary, on the part of our troops, was exaggerated
and made the most of, though what principally inflamed the
minds of the people were alleged instances of needless cruelty
which never occurred. Never in my life have I read of, much
less experienced, such a carnival of mendacity as that which
accompanied the pro-Boer agitation in this Colony at the end
of last year. And these libels still continue to make
themselves felt. …
"The present position of affairs, alike in the new territories

and in a large portion of the Cape Colony, if by no means the
most critical, is possibly the most puzzling that we have had
to confront since the beginning of the war. Naturally enough
the public are impatient, and those who are responsible for
the government of the country are bombarded with most
conflicting advice. On the one hand, there is the outcry for
greater severity and for a stricter administration of Martial
Law. On the other hand, there is the expression of the fear
that strict measures would only exasperate the people.
Personally, I am of the opinion, which I have always held,
that reasonable strictness is the proper attitude in the
presence of a grave national danger, and that exceptional
regulations for a time of invasion, the necessity of which
every man of sense can understand, if clearly explained and
firmly adhered to, are not only not incompatible with, but
actually conducive to, the avoidance of injustice and cruelty.
I am satisfied by experience that the majority of those Dutch
inhabitants of the Colony who sympathize with the Republics,
however little they may be able to resist giving active
expression to that sympathy, when the enemy actually appear
amongst them, do not desire to see their own districts
invaded, or to find themselves personally placed in the
awkward dilemma of choosing between high treason and an
unfriendly attitude to the men of their own race from beyond
the border. There are extremists who would like to see the
whole of the Cape Colony overrun. But the bulk of the farmers,
especially the substantial ones, are not of this mind. …
"The inherent vice, if I may say so, of almost all public

discussion of our South African difficulties is the tendency
to concentrate attention too exclusively upon the Boers. Say
what we will, the controversy always seems to relapse into the
old ruts—it is the British Government on the one hand, and the
Boers on the other. The question how a particular policy will
affect, not merely our enemies, but our now equally numerous
friends, seems seldom to be adequately considered. And yet it
would seem that justice and policy alike should lead us to be
as eager to consider the feelings and interests, and to retain
the loyalty, of those who are fighting on our side, as to
disarm the present enmity and win the future confidence of
those who are fighting against us. And this principle would
seem an the easier to adhere to because there is really
nothing which the great body of the South African loyalists
desire which it is not for the honour and advantage of the
Mother Country to insist upon. Of vindictiveness, or desire to
oppress the Afrikanders, there is, except in hasty utterances,
inevitable in the heat of the conflict, which have no
permanent significance, or in tirades which are wholly devoid
of influence, no sign whatever. The attitude of almost all
leading and representative men, and the general trend of
public feeling among the loyalists, even in the intensity of
the struggle, is dead against anything like racial
exclusiveness or domination. If this were not so, it would be
impossible for a section of pure bred Afrikanders, small no
doubt in numbers but weighty in character and position, to
take the strong line which they do in opposition to the views
of the majority of their own people, based as these are, and
as they know them to be, upon a misconception of our policy
and intentions. These men are among the most devoted adherents
to the Imperial cause, and would regard with more disfavour
and alarm than anyone the failure of the British nation to
carry out its avowed policy in the most complete manner. They
are absolutely convinced that the unquestioned establishment
of the British supremacy, and the creation of one political
system from Cape Town to the Zambesi, is, after all that has
happened, the only salvation for men of their own race, as
well as for others. Of the terms already offered, a great
majority, I believe, of the South Africans at present in arms
on our side entirely approve. There is, no doubt, an extreme
section who would advocate a sterner attitude on our part, but
they are not numerous, and their feelings are not lasting. The
terms offered by Lord Kitchener, which are, in substance,
identical with repeated declarations of policy on the part of
His Majesty's Government, are generally regarded as a generous
and statesmanlike offer, as one which, if firmly adhered to,
will ultimately be accepted, but as an offer which we cannot
afford to enlarge. On the other hand, there is a very general
desire that no effort should be spared to make the generous
character of our intentions widely known, and to encourage any
disposition on the part of the enemy to parley, with the object
of making them better acquainted with the terms on which we
are prepared to accept their submission.
"If I might sum up the predominant, indeed, the almost

unanimous feeling of those South Africans who sympathise with
the Imperial Government, I should describe it as follows:—They
are sick to death of the war, which has brought ruin to many of
them, and imposed considerable sacrifices on almost all. But
they would rather see the war continue for an indefinite time
than run the risk of any compromise which would leave even the
remotest chance of the recurrence of so terrible a scourge in
the future. They are prepared to fight and suffer on, in order
to make South Africa, indisputably and for ever, one country
under one flag, with one system of government, and that system
the British, which they believe to ensure the highest possible
degree of justice and freedom to men of all races.
{517}
But, with that object accomplished, they are willing, and,
indeed, ready, to bury racial animosities. They have fought
against the principle of race oligarchy in one form, and they
do not wish to re-establish it in another. For the attainment
of that object, they would rely for the present on the
vigorous prosecution of the war in which they are prepared
themselves to take the most active part, coupled with every
inducement to the enemy to come in on the terms already
offered, and for the future, as soon as public security is
assured and the circumstances permit, on the extension to the
newly acquired territories of a system of Colonial
self-government. For my own part, I have no doubt that this
attitude is a wise one, and that it only requires persistence
in it, in spite of the discouraging circumstances of the
moment, to lead us to ultimate success."
Great Britain, Papers by Command, Cd. 547.
The same Blue Book made known the fact that, on the 3d of
April, Sir Alfred Milner applied for and obtained leave of
absence for three months from his duties in South Africa.
SOUTH AFRICA: The Field of War: A. D. 1901 (April).

The situation.
Early in April it was announced that the seat of government of

the South African Republic had been transferred from
Pietersburg to Leydsdorp in the Zoutpansberg by the
Vice-President, General Schalk-Burger, which seems to indicate
the beginning of another stage of the South African war. The
Boers are said to have been for some time past collecting
great quantities of cattle and sheep in the fastnesses of the
Zoutpansberg, where also they have ample supplies of
ammunition, and intend making it a point of ultimate
resistance as well as a base of present operations.
SOUTH AFRICA: The Field of War: A. D. 1901 (April).

The cost of the war to Great Britain as stated
by the Chancellor of the Exchequer.
In his speech (April 18), on introducing the budget for 1901,

in the House of Commons, the Chancellor of the Exchequer, Sir
Michael Hicks-Beach, made the following statements of the cost
of the war to Great Britain: "I would remind the Committee
that so far we have borrowed towards the cost of the war
£67,000,000—£13,000,000 Treasury bills, £10,000,000 Exchequer
Bonds maturing rather less than three years hence, £14,000,000
Exchequer Bonds maturing about five years hence, and
£30,000,000 War Loan maturing in 1910. Now, Sir, in what mode
may we fairly borrow such a large sum as we now require? This
can no longer be considered a small war. In cost it is a great
war. Let me just make a statement to the Committee as to what,
so far, the estimated cost of this war has been. In 1899-1900 the
Estimates were £23,217,000. Last year they were £68,620,000,
and this year's Estimates amount to £60,230,000, including in
each case the interest on the sums borrowed. That amounts to
over £152,000,000. I must ask the Committee to remember that
in those figures I include the cost of both the South African
and Chinese wars. Then I have to add a million and a quarter
for this year's borrowing, making in all over £153,000,000.
That is double the cost of the Crimean War, and when I look
back at the Peninsular War I find the two most expensive years
were 1813 and 1814. The forces engaged, of course, were very
much smaller than those engaged now; but in those two years
the total cost of our Army and Navy amounted to £144,581,000.
This amount is less than the charges of the South African and
Chinese wars. Therefore, I think I am justified in saying that
in cost this has been a great war. I think, then, it is clear
we can no longer, in borrowing towards the cost of it, rely
upon temporary borrowing. We have already £67,000,000 of
unfunded debt borrowed for this purpose and maturing within
the next ten years. We have also some £36,000,000 of 2¾ and 2½
per cent., redeemable in 1905. Therefore, whatever may be the
prosperity of the country, whatever may be the condition of
our finances, it is perfectly obvious to my mind that the
stanchest advocate of the redemption of the debt will have
ample scope for his energies in the years that are now before
us. For this reason I propose to ask the Committee to extend
the powers of borrowing which they gave me in previous Acts,
to Consols."
----------SOUTH AFRICA: End--------
SOUTH AFRICAN REPUBLIC, The.
See (in this volume)

SOUTH AFRICA (THE TRANSVAAL);
also,
CONSTITUTION (GRONDWET) OF THE SOUTH AFRICAN
REPUBLIC.
SOUTH AUSTRALIA.
See (in this volume)

AUSTRALIA; and CONSTITUTION OF AUSTRALIA.
SOUTH CAROLINA: A. D. 1892-1899.

The Dispensary Law.
In 1892 the Legislature of South Carolina passed an Act,

commonly called the Dispensary Law, which caused turbulent
agitations in the State, and excited much interest in the
country at large. It was based upon the principle of what is
known as the Gothenburg system of regulation for the sale of
intoxicating liquors, making the traffic a State monopoly,
carried on by officials, under rigorous restrictions, with
profit to the public treasury, and none else. It provided for
the creation of a State Board of Control, under the direction
of which a Commissioner, appointed by the Governor, should
purchase all intoxicating liquors allowed to be sold in the
State, and should furnish the same to such agents (called
"dispensers") in the several counties as might be appointed by
county boards to sell them, in accordance with the regulations
prescribed. It required all liquors purchased by the
Commissioner to be tested by an official chemist and declared
to be pure and unadulterated. It allowed nobody but the
official "dispensers" to deal in any manner with any kinds of
intoxicating liquors after the 1st of July, 1893. It forbade
the selling of such drinks by the authorized salesmen to
minors and drunkards, and it required all who bought to sign
and date a printed or written request, stating their residence
and age.
The law was fiercely resisted in many parts of the State by

mobs, and powerfully assailed in the courts; but Governor
(afterwards Senator) Tillman, who then occupied the executive
chair, gave it resolute enforcement and support. The attack in
the courts had momentary success in 1894, the Supreme Court of
the State rendering a decision adverse to the
constitutionality of the law; but, meantime, the Legislature,
in 1893, had made changes in the Act, and its new enactment
was held to be untouched by the judgment of the court.
{518}
Before a new case could be brought to issue, the retirement of
one of the justices of the Supreme Court brought about a
change of opinion in that tribunal, and the law in its new
form was sustained. Disorderly resistance to the enforcement
of the law was long kept up; but in the end such resistance
seems to have been mostly overcome.

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

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

an Fc fragment, it is hoped a similar effect of increased circulation time will be achieved. As

1.4.4.2 Example 2: PEGylated proteins and antibody drug conjugates (ADCs)

1.4.4.3 Example 3: viruses, VLPs

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

I. Proteins and biophysical characterization in the biopharmaceutical industry

[62] Quan S, Bardwell JC. Chaperone discovery. Bioessays 2012;34:973e81.

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

Out of the instructions he received, Lord Kitchener finally

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