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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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The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

Copyright © 2020 Elsevier B.V. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
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This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).

Notices
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understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
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 Contributors

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

xi
 Prefaces for the second edition

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

successful therapeutic drug is our ability to tion that there was room for improvements.
identify and manufacture the drug such As a result, in writing this second edition
that its beneficial therapeutic effect can be we have undertaken the job of updating old
safely delivered to the patient. In the case of information, correcting mistakes, improving
protein biopharmaceuticals, these large, clarity and the introduction of new topics
heterogeneous (complex) and marginally that were not covered in the first edition.
stable molecules are often very sensitive to Therefore, we gathered our co-authors once
their micro-environment. This makes the again, invited a few new ones, and tasked
process of developing and manufacturing a ourselves with the goal to achieve these
protein therapeutic extremely challenging. objectives. In so doing, all original chapters
Throughout this entire process a protein bio- have been updated, corrected and
pharmaceutical must maintain its complex enhanced, while new chapters have been
and delicate structure (or conformation) to added.
realize its beneficial therapeutic attributes, Globally, the format of the book has
while avoiding the potential harmful effects remained the same, consisting of three sec-
in failing to achieve this goal. tions. Section I, which deals with the
When we set out to write the first edition complexity of proteins and the relevance of
of this book, our goal was to provide a biophysical methods in the biopharmaceuti-
general resource that specifically dealt with cal industry. It has for the most part been
the many challenges associated with the altered to remove errors and achieve clarity.
testing and characterization of the higher Section II, which discusses the biophysical
order structure and biophysical properties of tools and techniques most commonly used in
protein biopharmaceuticals from a practical the biopharmaceutical industry to charac-
point of view to support its safety and terize protein therapeutic molecules has
beneficial therapeutic activity. As stated in similarly been altered, but has also been
the book’s first preface we wanted to keep enhanced by the addition of a new chapter
the reader focused on obtaining a pragmatic (Chapter 14) dedicated to the area of chro-
understanding and knowledge of the utility matography and electrophoresis. The tools in
of biophysical tools and how they are used to this chapter, which we did not cover in the
meet these challenges by understanding first edition of the book (with the exception of
what information can realistically be extrac- size-exclusion chromatography), are typically
ted from these tools. While we felt we had not thought of or classified as biophysical
initially achieved our goal, the progression of tools. Nevertheless, an important objective in
time inevitably led to better and improved adding this chapter is to bring more attention

xiii
xiv PREFACES FOR THE SECOND EDITION

to their unrealized linkage as effective bio- Finally, we would like to point out that
physical characterization tools without get- in writing this second edition we have
ting too deep into the details of their inner made a particular effort, wherever possible,
workings (which are extensively covered in to better link and cross-reference informa-
many excellent books and review articles that tion in each chapter to bring more cohesion
are solely dedicated to these two enormously to the book as appose to just providing
important techniques). the reader with a collection of isolated
Overall, however, Section III of the book chapters. We think his cohesion is in partic-
has experienced the most significant change ular made apparent by the four additional
and expansion via the addition of four new chapters in Section III (described above).
chapters that cover the following: In the end, we and our coauthors hope we
have further enhanced the initial objective of
• Chapter 15, which deals with the biophysical
characterization of complex biopharmaceuticals; the first edition of the book, of enlightening
the reader to the challenges, tools and inner
• Chapter 16, which deals with the rigor of
workings of the task associated with the
statistical analysis;
biophysical characterization of protein bio-
• Chapter 17, which deals with biopharmaceu-
pharmaceuticals. An integral part of today’s
tical developability;
modern and challenging world of devel-
• Chapter 18, which deals with technical deci-
oping lifesaving drugs.
sion making.
 List of abbreviations and symbols

(T)RPS (Tunable) resistive pulse sensing

3D Three dimensional
A22 or B2 Second viral coefficient
AAV Adeno-associated virus
AC Alternating current
AC-SINS Affinity-capture self-interaction nanoparticle spectroscopy
AFFF-MALLS Asymmetric field flow fractionation with multi-angle laser light scattering
ACN Acetonitrile
ACS Ammonium camphor sulfonate
ADC or ADCs Analog to digital converter or Antibody drug conjugate(s)
ADCC Antibody dependent cell-mediated cytotoxicity
AF4 Asymmetric flow field flow fractionation
AFM Atomic force microscopy
API Active pharmaceutical ingredient
APR Aggregation prone regions
AQL Acceptable quality level
ASTM American society for testing and materials
ATP Analytical target profile
ATR Attenuated total reflectance
AUC Analytical ultracentrifugation
BiAb or bsAb Bispecific antibody
BLA Biological license application
BMI Backgrounded membrane imaging
BSA Bovine serum albumin
CA Capsid protein
CAD Collision-activated dissociation
CCD Charge-coupled device
CD Circular dichroism
CDER Center for drug evaluation and research
CDR Complementarity-determining region
CEX Cation-exchange chromatography
cGMP Current good manufacturing practices
CH Immunoglobulin gamma heavy chain constant domain
CH1 or CH1 Immunoglobulin gamma heavy chain constant domain 1
CH2 or CH2 Immunoglobulin gamma heavy chain constant domain 2
CH3 or CH3 Immunoglobulin gamma heavy chain constant domain 3
CHO Chinese hamster ovary
CIC Cross-interaction chromatography
CID Collision induced dissociation

xv
xvi LIST OF ABBREVIATIONS AND SYMBOLS

cIEF Capillary isoelectric focusing

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

LMW Low molecular weight

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

TDA Taylor dispersion analysis

TEM Transmission electron microscopy
TIC Total ion current
TM-DSC or MT-DSC Temperature-modulated differential scanning calorimetry
TMU Target measurement uncertainty
TPP Target product profile
Try Tyrosine
TSP Trimethylsilyl propionate
Tyr Tryptophan
UPLC or UHPLC Ultrahigh performance liquid chromatography or ultra-performance liquid
chromatography
USP United States Pharmacopeia
UV Ultraviolet light
UVeVIS Ultravioletevisible spectroscopy
VH Immunoglobulin gamma heavy chain variable domain
VIS Visible light
VL Immunoglobulin gamma light chain variable domain
VLP or VLPs Virus-like particle(s)
WAXS Wide angle X-ray scattering
WCX Weak-cation exchange chromatography
WSD Weighted spectral difference
XIC Extracted ion chromatogram
Z Net charge
 C H A P T E R

1
The complexity of protein structure
and the challenges it poses in
developing biopharmaceuticals
Steven A. Berkowitza, Damian J. Houdeb
a
Consultant, Sudbury, MA, United States; bBiomolecular Discovery, Relay Therapeutics,
Cambridge, MA, United States

1.1 The basics of protein higher order structure (HOS)

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

Biophysical Characterization of Proteins in Developing

Biopharmaceuticals, Second Edition 3
https://doi.org/10.1016/B978-0-444-64173-1.00001-9 Copyright © 2020 Elsevier B.V. All rights reserved.
4 1. The complexity of protein structure and the challenges it poses in developing biopharmaceuticals

FIG. 1.1 (A) The linear sequential ordering of amino acids (represented by the rectangular black dashed boxes) in
a protein is referred to as its primary structure. The extreme left amino acid corresponds to the amino-terminus, while
the extreme right amino acid corresponds to the carboxyl-terminus end of the protein chain. The gray shaded area
corresponds to the peptide bonds that link all the amino acid units in a protein, yielding the polypeptide backbone (or
chain) indicated by the red (gray in print version) dotted rectangle. (B) An illustration of the planar structure of two
adjacent amide planes (each resulting from the double bond character, due to resonance, of the peptide bond shown
as black dashes), corresponding to the light blue (light gray in print version) shaded areas in (A), where the bottom
amide plane is formed from the peptide bond between the carboxyl group of amino acid 1 (containing R1) and the
amino group of amino acid 2 (containing R2) and the top amide plane is formed from the peptide bond formed
between the carboxyl group of amino acid 2 and the amino group of amino acid 3 (containing R3). Due to steric issues,
angular rotation around CaN (expressed by F, phi) and CCa (expressed by J, psi) bonds are limited. (C) A rep-
resentation of a common secondary structure, the a-helix. The small rectangle outlined in black dashes corresponds to
a small section of the helical arrangements of the amide planes, shown in (B).

staggering number of different possible proteins, 20N, to be made. Given the enormous array
of different proteins that can be made, the cell has exploited this diversity in protein structure
to create proteins to perform nearly every functional and structural role needed for its
existence.

I. Proteins and biophysical characterization in the biopharmaceutical industry

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

1.1.1 The levels of protein HOS

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

I. Proteins and biophysical characterization in the biopharmaceutical industry

6 1. The complexity of protein structure and the challenges it poses in developing biopharmaceuticals

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

I. Proteins and biophysical characterization in the biopharmaceutical industry

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