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Protein aggregation: current scenario and recent developments

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Protein aggregation: current scenario and recent developments

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PROTEIN AGGREGATION: CURRENT SCENARIO AND RECENT
DEVELOPMENTS

by

Manali Shah

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2012

Copyright 2012 Manali Shah
ii

ACKNOWLEDGMENTS

I would like to show my utmost gratitude towards my Advisor and Chair, Dr.
Wei-Chiang Shen who has always supported me in all my endeavors. His
belief in me and my work has always proved an inspiration to explore new
horizons and bring about new and creative ideas. He is a great mentor who
taught me how to sail through difficult times and emerge as a successful
person.
Thank you Sir, for your constant support and guidance!
I would also like to thank my fellow lab members who have been very
helpful and kind and made the whole journey of my graduate study in USC
a memorable one.
My heartfelt appreciation goes towards my family and a very special thank
you to all my friends here in USC who stayed with me during good and bad
times and helped me put up my thesis together.

Thank you guys, you all made “USC” an unforgettable and amazing
experience
iii

TABLE OF CONTENTS

Acknowledgments ii
List of Tables iv
List of Figures v
Abstract vi
Chapter 1: Introduction 1
Chapter 2: Mechanisms of Aggregation 5
Chapter 3: Nature and Extent of Aggregation 17
Chapter 4: Techniques to Detect Aggregation 29
i. Common Strategies 30
ii. Nuclear magnetic Resonance 34
iii. X-Ray Fiber Diffraction 37
iv. Dynamic Light Scattering Microscopy 39

Chapter 5: Inhibition of Protein Aggregation 41
i. Common Strategies 41
ii. Specific Approaches 44

Chapter 6: Storage of Proteins 53
Chapter 7: Toxicities/Diseases: A result of protein aggregation 55
i. Alzheimer’s Disease 57
ii. Prion Disease 67

Chapter 8: Conclusion 74
References 77
Comprehensive References 82
iv

LIST OF TABLES
Table 1: Analytical methods used for protein detection 31

Table 2: Overview of analytical methods for detection of 32
protein aggregation based on structure, folding and assembly

Table 3: Approaches towards inhibition of protein aggregation 43

Table 4: Summary of therapeutic strategies for protein aggregation 64
diseases

v

LIST OF FIGURES
Figure 1: Reversible association of protein aggregates 7

Figure 2: Oligomerization following conformational change 9

Figure 3: Oligomerization driven by covalent modification 11

Figure 4: Nucleation controlled seeding mechanism 13

Figure 5: Surface induced aggregation 14

Figure 6: Aggregation behavior and detection signals 35
using Nuclear Magnetic Resonance Spectroscopy

Figure 7: Electron microscope image of amyloid fibrils 37
by negative staining of IAPP

Figure 8: Stained section of Human cortical brain of Alzheimer’s patient 60

Figure 9: Three-dimensional ribbon diagram of Aβ-amyloid protein in 62
cross β-spine conformation

Figure 10: Proposed mechanism of Amyloid-β formation 63

Figure 11: Stained sections of human cortical brain of Prions’ 69
disease diagnosed patient

Figure 12: SDS-PAGE of PrP-specific brain homogenates 70
digested by proteinase K

Figure 13: Proposed mechanism of PrP
c
mediated oligomer 72
formation and initiation of toxicity

vi

ABSTRACT

Aggregation of proteins is the major topic of discussion and debate in
the biopharmaceutical industry. Various chemical and physical properties of
proteins have been studied and manipulated in order to overcome this
major hurdle during biopharmaceutical product formulation. Huge
investments are done in order to understand the mechanisms underlying
abnormal protein aggregation during the production process as well as in-
vivo. Theories explaining this aggregation process are manipulated in the
industry in order to attain the protein drug in its highest quality. Further,
based on the nature and extent of such aggregates formed it is possible to
classify the different types of aggregates. As aggregation is assumed to be
an end-product of undesirable events occurring through the molecular
cascade, based on such classification one can try to reach to the possible
causative factor in the molecular cascade that leads to aggregation. Various
detection techniques have been utilized to check the presence of aggregate
formation while the protein drug is in its initial processing steps. Such
techniques provide a lot of information on physical characteristics and real
vii

time detection on how the process of aggregation moves in its pathway.
Further, we have discussed the most widely used techniques and strategies
to inhibit aggregation at the in-vitro level. Storage of proteins in order to
maintain it viable for a sufficiently long interval of time is very important.
Techniques on effective storage and environmental conditions that may
affect as a causative parameter in initiation of aggregation have been
discussed. Taking a step further in this direction, many neurodegenerative
disorders have found their origins in the formation of protein aggregates,
called amyloids. We have discussed two major neurodegenerative diseases:
Alzheimer’s disease and Prion Disease and their pathogenesis.

1

CHAPTER 1
Introduction
The past couple of decades showed an explosive interest in the
biopharmaceutical industry and a majority of discoveries using proteins as a
treatment strategy were introduced. However, in spite of this huge amount
of interest in biopharmaceutical products, an equal amount of
commercialization and widespread use as an actual drug for clinical
treatment has not been achieved. Protein aggregation, the most common
and problematic manifestation encountered in the various stages of protein
drug development sums up to be a majority of the various instabilities of
the protein along with physical and chemical instabilities that prove to be a
consequential barrier in the commercialization of protein drugs as
candidates of new treatment therapies. Not only aggregation of proteins
but also the presence of any insoluble particulate matter or impurity is
unacceptable in any protein formulation. Abnormal protein aggregation has
proved to be a root cause for various diseases which result in physical or
chemical changes in the cellular environment and sometimes may affect
2

the DNA and cause mutations in the gene sequence, hence rendering toxic
to the cellular body
1
. Neurodegenerative diseases such as Alzheimer’s
disease (AD), Huntington’s disease (HD) Parkinson’s disease, amyotrophic
lateral sclerosis (ALS) and Prion diseases have shown evidence of similar
pathogenesis with respect to common mechanisms at cellular as well as
molecular level including protein aggregation and inclusion body
formation
2
. These aggregates are identified as fibers incorporating a β-
sheet conformation containing misfolded proteins. Such misfolded proteins
are termed as amyloid. These abnormal proteins get deposited on cell
surfaces and lead to cell degeneration. The most convincing explanation of
this deposition is that the inclusions and aggregates get detected in the end
of the molecular cascade rather than the initial steps and hence the earlier
steps in the cascade would be more effective in diagnosing the
pathogenesis of the diseased state. Other possible reason can be termed to
genetic variations thus explaining about the pathogenesis of most common
sporadic forms and mouse models. Proteins posing for a possible risk of
neurodegenerative disease are likely to be natively unfolded. There are
3

several kinds of protein aggregates of which amyloid fibrils are the most
characteristic. The question to answer here is what might have caused this
aggregation process. A lot of interest is now focused on understanding
these pathways and molecular cascades which prove to be a savior for the
emerging biopharmaceutical products and provide new insights on
prevention and mitigation of protein aggregation and carve a new path
towards rational biopharmaceutical therapeutics. Also, majority of the
resources in the pharmaceutical production industry are spent on
developing ways to prevent protein aggregation by early detection and
possible removal of any aggregate in their product or formulation. In this
thesis, I will try to go over some major issues that come across the
development of a biopharmaceutical process. I will discuss in detail about
the proposed mechanisms of protein aggregation and theoretical concepts
that have been used lately in order to understand the protein aggregation
process. Further, I will introduce the reader towards different nature of
aggregates and their extent of aggregation. With the advancement in
science and technology, a variety of tools and techniques that are used to
4

detect possible aggregates in the production process will be discussed.
Proposed mechanisms to prevent and mitigate aggregate formation will be
discussed in detail. Further, strategies used for effective storage of proteins
for long intervals of time are reviewed. Finally, consequences and in-depth
review of such an aggregation process auto-initiated in the body of an
individual will be illustrated using the example of Alzheimer’s disease and
Prion disease.

5

CHAPTER 2
Mechanisms of aggregation
Protein aggregation is detected usually at the end of the molecular
cascade and proves to be detrimental to the final product. However,
various theories have been proposed to explain the flow of steps that may
result into a protein being rendered biologically inactive and a possible risk
to the final product. Various mechanisms are proposed which may play an
important role in the cascade of events of protein aggregation, some of
which are described as below:
1. Reversible Association of Native Protein
3

Reversible self association of monoclonal antibodies has
shown to effect mainly by two parameters: electrostatic interactions
of charged residues or surface dipole moments of the proteins. These
low affinity and multivalent interactions might lead to high degrees
of protein aggregation and ultimately an unusual high viscosity of
highly concentrated antibodies in low ionic strengths buffers. Despite
reversible nature such an association can have a major impact on
6

important pharmaceutical properties. On oligomerization, they may
remain in the same conformation or have a slightly different
conformation. The proposed mechanism is based on the studies
carried out on concentrated monoclonal antibodies in aqueous
solutions. A reversible self association, as per the theory suggests,
could be a possible cause of the observed increased viscosity of the
aqueous solution. The viscosity of monoclonal antibodies (MAb1)
solutions depends on pH under hypotonic conditions. The theoretical
pI of the protein is attained approximately at neutral pH and
viscosity changes are observed under low salt conditions. Moreover,
there is a decrease in viscosity observed with respect to the ionic
strength of the solution. This suggests that electrostatic interactions
contribute to increase in viscosity and are a major cause for protein
aggregation in aqueous solutions of MAb1.

7

Figure 1: Reversible association of protein aggregates

2. Oligomerization following conformational change
4

Multimerization highly affects the high affinity binding of Heat
shock factor (HSF) to DNA. However, the exact quaternary structure
of the HSF activated form has not yet been resolved. Studies
involving S. cerevisiae show that HSF protein is present as a trimer of
identical subunits in an aqueous solution. Heat shock activated form
of Drosophila HSF also showed a similar trimeric association from
chemical cross linking experiments. HSF expressed in E.Coli shows a
Protein monomer
Reversible oligomer formation
8

collection of trimers, hexamers and higher multimers in the sample.
Further, using fluorescence resonance energy transfer (FRET) analysis
the formation of oligomers and conformational changes in the
Na+/H+ antiporter from Helicobacter pylori (HPNhaA) were studied.
Proposed mechanism involves an initial conformational change
converting the native protein into a modified protein which then
readily undergoes oligomerization and initiates further
conformational changes. The aggregates formed after
conformational changes are believed to be dissociable in nature.

9

Figure 2: Oligomerization following conformational change

Conformational Change
Oligomer formation: Altered
conformation, dissociable
Protein monomer
10

3. Oligomerization driven by covalent modification
4,5

Each individual protein molecules in an aggregate are exposed
to chemical modifications. Such interactions and modifications
include cross linking between amino acids or modification of residue
units. Irreversible aggregates are a result of cross linking between
amino acids, particularly presence of covalent cross linking. Covalent
cross linking majorly involves disulphide cross-links, which are
reducible. These cross links include intermolecular cross linking and
intramolecular modification of protein moieties. Non reducible
chemical cross links like thioether and dityrosine are present in
protein aggregates. Other crosslinking events can be as a result of
various chemical reactions like oxidation of cysteines or methionines,
deamidation etc which also play an important role in protein
aggregation.

11

Figure 3: Oligomerization driven by covalent modification

?
?
Protein monomer Conformational Change
Covalent linkage
12

4. Nucleation controlled aggregation (Seeding)
6

This mechanism proposes that once a critical nucleus with n
number of monomers is formed, presence of additional monomer
units surrounding the nucleus provoke association and formation of
larger aggregates. A certain delay time, called the lag period is
required for the nuclei formation. Presence of buffer affects the size
of nuclei formation. As demonstrated in studies carried out in
investigating the aggregation mechanism in Deoxyhemoglobin S, the
size of nuclei produced in a concentrated phosphate buffer solution
is smaller than that formed when gelation of deoxy-Hb S occurs in
low phosphate buffer solutions. Studies carried out by Hofrichter et
al reveal that the initial size of the nucleus is determined by the
super saturation and is a result of the competition between bonding
free energy between the two monomers and net gain in rotational
and translational free energy gained through aggregation. The results
reported from study of deoxy-Hb S suggest that the aggregates have
a negative temperature coefficient and can be easily melted by
13

cooling or bubbling with carbon monoxide gas. However, the kinetics
of reaggregation is affected by the duration of cooling time and that
the polymers of deoxy-Hg nuclei dissociates slowly into the buffer
media.

Figure 4: Nucleation controlled seeding mechanism

+
Native Protein
Critical Nucleus
Addition of protein
monomers onto the
surface of nucleus
Visible aggregates or precipitation
14

5. Surface Induced aggregation
7

Figure 5: Surface induced aggregation

Native
protein
Container
surface and gas-
liquid interface
Adsorption of
monomers on
surface and
partial unfolding
Aggregation of
altered protein
15

Studies carried out for demonstration of adsorption of ferritin
on a hydrophobic surface very well depicts the concept of surface
induced aggregation mechanism of the proteins. The adsorption of
ferritin on a hydrophobic surface was studied using transmission
electron microscopy. Molecular clusters were detected as a
distribution pattern of ferritin on the surface. Adsorption process
showed diffusion as the rate limiting step after a 20 hour adsorption
time at concentrations below 1mg/L. These studies demonstrate the
strong dependency of the supramolecular structure of the adsorbed
protein layer to the kinetics of ferritin adsorption and that a stable
plateau level of adsorption is present below a monolayer. The study
elaborates on effects of concentration changes on the plateau level
after a long period of adsorption. The electron micrographic images
of ferritin adsorbed to a hydrophobic quartz surface from different
concentrations of bulk solutions at an interval of 20hr were
performed. Small and stringy clusters were observed within a short
time after fixation with glutaraldehyde. Such small clusters have a
16

tendency to condense and become confluent when adsorbed from a
higher bulk concentration (≥1 g/L) and are sensitive to rinsing. In
cases of higher bulk concentrations where the adsorption becomes a
reaction rate limiting process, the aggregation rate at the surface is
very high. The rate of dissociation may then increase with the
increase in the coordination number of the aggregates and hence
result in a structure dependent dynamic equilibrium. This concept is
in good standing with other experimental derivations that there is a
constant and detectable exchange of proteins in the solution and on
the adsorbed surface compared to those proteins which are
irreversibly bound to surface when in salt solutions.

17

CHAPTER 3
NATURE AND EXTENT OF AGGREGATION
The process of protein aggregation is quite complicated and
depends on a variety of factors like solvent conditions, amino acid
sequence, sample history, pH, presence of suitable buffer,
electrostatic interactions and so on. Our ability to diversify each
factor and understand it individually depends on identifying the vast
category of aggregates that form during the course of
biopharmaceutical product formation. In order to identify in detail
about the nature and extent of our protein aggregates, the following
five categories have been proposed by Narhi, et al
8
:
1. Size
2. Reversibility/dissociability
3. Conformation
4. Chemical Modification
5. Morphology

18

1. Size
Narhi et al. described the term oligomer as: “Oligomers
can be defined as any aggregate that contain a few monomeric
units, in keeping with the International Union of Pure and
Applied Chemistry (IUPAC) definition; these would generally be
in the submicron size range depending on the monomer”.
Using quantitative categories like <100nm, 100-1000nm (sub
micrometer), 1-100 micrometer, >100 micrometer is more
useful when a detectable and measureable aggregation rate
has to be determined. Size classification is the best and most
widely used characteristic for identification and classification
of aggregates, however using subjective terms like visible/sub
visible or soluble/insoluble has created quite some confusion
in its usefulness. While providing a quantitative characteristic
to the aggregates, the technique used to determine it may
have a profound effect on the apparent size distribution of the
observed values.
19

2. Reversibility/Dissociation
3,8

Self-association of proteins leads to either reversible or
irreversible association between monomer units. This is a very
important property as to how the proteins undergo self-
association between the species. The term “reversible” can be
coined to those aggregates which are in thermodynamic
equilibrium with the monomeric units in the solution under
specific conditions. Also, dissociation of these aggregates may
be detected on experimental scale. Reversible aggregation is a
characteristic of relatively weak non-covalent protein
interactions and can be explained thermodynamically.
Dissociation in such aggregates can be achieved by dilution of
the solution. In case of irreversible aggregation, the aggregates
formed are higher molecular weight species and which cannot
be reversed back into the solution with use of reducing agents
or denaturants or other stressors. Separation can be achieved
by chromatographic isolation. When the proteins are
20

reinjected into the column they will elute in the same position.
This technique can also be used as a means of purification.
However, if the dissociation of the aggregates follows a
prohibitively long scale it is still possible that aggregates can be
solubilized through use of conditions like higher temperature,
buffers or building up special circumstances beyond the simple
steps that led to the formation of aggregates. There is one
more kind of aggregates called the dissociable aggregates.
Dissociable aggregates are non-reversible but still can be
recovered by manipulating the solution conditions like change
of temperature, pressure or other physiological conditions.
On the whole, a combination of all the three kinds;
namely reversible, irreversible and dissociable kinds are found
in protein aggregate samples.

21

3. Conformation
1, 4, 8

A change in conformation of the protein gives a basis for
classifying them under the following categories
8
:
a) Native
b) Partially unfolded
c) Misfolded
d) Inherently disordered
e) Unfolded
f) Amyloid
Moreover, parameters like stability, surface
hydrophobicity are also considered as identifiers of the
conformational change of protein molecules.
As the name suggests, “native” conformation identifies
the original conformation of the protein when produced
through engineering or extracted from biological processes. In
this state the protein is active and retains all its characteristics.
22

“Partially unfolded” and “unfolded states” refer to those
changes in the conformation of the protein that can be
detected via analytical methods. However, these states also
retain some properties of their native conformation.
“Misfolded” states refer to those aggregates which show
a completely different folding arrangement within the protein
and have characteristic difference in physical properties. These
proteins do not retain biological activity and are rendered as
impurities or potential toxins in the product or formulations.
For example, in the production of a protein from a biological
organism, there may be changes in the shape or conformation
of the beta sheet which is originally supposed to be α-helical in
shape.
“Unfolded” state refers to that conformation of the
protein which can be compared to the conformation of an
artificially denatured protein sample using extreme
23

environmental conditions like 6M Guanidine hydrochloride
and rendered denatured and aggregated.
“Inherently denatured” state refers to those aggregates
whose conformation resembles the conformation of synuclein,
amyloidal-β and other disordered proteins which lack the basic
amyloid signature of 4.7Å and 1nm cross-β diffraction
pattern.
8, 9

4. Chemical modification
8-11

Presence and formation of chemical bonds between
amino acids can lead to major changes in the shape,
conformation and biological activity of the protein. Individual
monomers can undergo chemical cross linking between
successive amino acids in the sequence or interlinks between
residue moieties. The type and strength of such a chemical
modification plays a vast role in the development of a
reversible or an irreversible aggregate. Covalent linkage
24

between monomeric units can lead to irreversible aggregate
formation which can be further classified into either inter-
molecular cross linking or intra-molecular modification
8
. More
variation in covalent linkage includes the formation of
dityrosine and thioether bond formation. Certain redox
conditions, chemical reactions like deamidation, reduction,
and oxidation or amino acid moieties of cysteine or
methionine have contributed to the majority of the reasons for
protein aggregation
10
. These reactions in itself provide the
explanation of the possible mechanism of action in the process
of aggregate formation during production and formulation
process.
Certain parameters like pH of the buffer, redox
conditions, temperature of the reaction, and presence of
byproducts during the reaction or addition of solubilizing
agents are widely studied in the formation of drugs and other
chemical entities. However, limitations are experienced using
25

these same conditions while using proteins or other biological
product as our reactant. Modifications in formulation steps
need to be made in order to maintain our protein biologically
active and sustain all its physicochemical properties.

5. Morphology
1, 8, 12

In case of dimmers, trimers or other higher oligomers it
is very important to specify the morphology of the proteins in
the sample. If known, the array of the monomers must be
specified in order to reduce the chances of confusion between
protein aggregates with oligomers and other non-
proteinaceous substances that may have entered the product
sample. In brief, studying the morphology of the protein can
serve as a very important tool in order to differentiate, at later
stages of the formulation and production, the different kinds
of aggregates, mechanism of aggregation and identify the
presence of intrinsic and extrinsic foreign particles. Intrinsic
26

and extrinsic particles render to protein impurities and can
serve as nucleation or instigating points for aggregation to
occur. Extrinsic particles are those that may have entered the
production batch during processes of cleaning, assembly
setup, insect parts, paint flakes, dirt or grease from the
machine parts and tubing etc. Intrinsic particles can be
described as those that can enter the formulation if filtration
was not carried out in strict control of the validated procedure
and some residue might enter the filtrate. Further, lubricant
oils used in medical delivery devices, for e.g. Silicone oil can
enter the product during the flow of solution from different
devices or machines
13
. Certain characteristic parameters are
identified which help in identifying these aspects with relative
ease and simplicity of application. Such parameters and their
possible applications are as discussed below
8
:
a) Aspect Ratio
b) Surface Roughness
27

c) Regular/amorphous structure
d) Shape : Fiber/Sphere
e) Optical properties : Refractive index and transparency
For a solution of protein, measurement of the above
mentioned parameters serve as an important tool in
determining the purity of the sample and hence detect
presence of any possible aggregation. The major advantage of
such parameters is that these observations can be carried out
at any possible time point during the production process. Early
detection of any possible impurity will serve as the most
crucial and positive advantage of a biopharmaceutical product
evaluation. Such techniques are cost effective and in the long
run will serve as the most important procedures while
investing large capital amounts in a new biopharmaceutical
product formulation.
Optical properties are characteristic of the protein
molecule and can be exploited in detection of presence of
28

aggregates and other extrinsic and intrinsic foreign materials.
Also, it serves as a differentiating factor for detection between
aggregates, oil droplets, air bubbles or other non-
proteinaceous substances. However, many proteins have
similar refractive indices and density and hence differentiating
between such native proteins and their aggregates become a
difficult task. Further, the concept of heterogonous protein
aggregates must also be introduced in this discussion which
can possibly escape the detection methods and serve as a false
positive result. Such heterogeneous aggregates include those
aggregates that have been formed due to the incorporation of
non-protein contaminants into the therapeutic protein.
Examples of non-protein contaminants include glass, fibers,
dust particles, grease, stainless steel particles etc. Such
aggregates alter the solvent content, fractal dimensions and
packing geometry of the proteins while incorporated in the
aggregate form
13
.
29

CHAPTER 4
TECHNIQUES TO DETECT AGGREGATION

Amyloid deposits, more commonly known as protein aggregates,
have similar structural characteristics but the individual monomers
comprising the amyloid protein show wide differences in their primary
structures
14
. Hence, a lot of investigations are being made in order to
decipher the structure, arrangement and assembly of amyloid proteins and
their precursors.
Amyloids that result in a diseased state in the individual are generally
classified into two major categories
14
:
1. Globular proteins
2. Natively unfolded proteins
1. Globular proteins: These classes of amyloidal proteins require partial
unfolding as the initiation step towards aggregate formation, further
leading to an unstable intermediate conformation which ultimately self-
associates into toxic aggregates that are rich in β-sheets
15
.
30

Example: Prion, Transthyretin (TTR), Cu/Zn superoxide dismutase 1 and
β
2
micro globulin.
2. Natively unfolded proteins
15

These proteins have a similar mechanism of aggregation as the globular
protein the only difference being that they are mainly composed of
amyloid β-protein (Aβ).
Example: Α-synuclein, tau protein, amyloid β-protein

I. Common strategies for detection of aggregates

Table 1 describes the categorization according to the analytical methods
used to detect protein aggregation whereas Table 2 describes
categorization of analytical methods depending on their structure,
folding and assembly.

31

Table 1: Analytical methods used for protein detection
Ref: Wei Wang. Protein aggregation and its inhibition in biopharmaceutics; International
Journal of Pharmaceutics 289 (2005) 1-30
32

Among all these methods listed above the most simple and effective way of
detection of protein aggregation is through visual inspection. Aggregates
are easily detected under a high powered microscope or even more
Table 2: Overview of analytical methods for detection of protein
aggregation based on structure, folding and assembly
Ref: Huiyuan Li et al., Amyloids and protein aggregation-Analytical methods,
Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) Copyright © John Wiley and
Sons Ltd

33

sophisticated electron microscopes for smaller aggregates and a high
power resolution. Furthermore, using turbidity as the means of studying
extent of aggregation is a very traditional approach and obtained by
measuring the optical density of the solution. Principles of light scattering
and spectroscopy are involved in such techniques. A large range of
wavelengths have been utilized for detection of protein aggregates via UV
or visible spectroscopy. For e.g. detection of urokinase was done using
300nm
16
, aFGF or insulin using 350nm
17
.Such methods have an important
assumption to make that the aggregates are uniformly distributed
throughout the protein solution, which may not be the case in most of the
sample batches. More often, aggregates are found stuck to the bottom of
the vials or any other surface or corners of the solution. Hence, such
methods have a limitation of measurements only to a relative degree and
extent of protein aggregation and cannot determine the actual
quantification id the aggregates in the solution.

34

We discuss in detail the following analytical techniques:
II. Nuclear Magnetic Resonance (NMR)
This analytical method has an advantage of detecting protein
aggregates along with atomic resolution and determination of a
three dimensional structure of the protein complexes.
The mechanism of action of NMR involves the use of magnetic field
of the inherent nuclei of the atoms comprising of the protein
structure. When subjected to high magnitudes of magnetic field,
these nuclei undergo changes in their spin quantum giving rise to a
signal that is detected by the detector and recorded as a peak. In
biopharmaceuticals, the nuclei that can be studied are
1
H,
15
N,
19
F
and
31
P. The following graph depicts the formation of aggregates in
Amyloid β protein with respect to time after sample preparation
using solution-state NMR analytical method.

35

Figure 6: Aggregation behavior and detection signals using Nuclear
Magnetic Resonance Spectroscopy
Aggregation behavior of Aβ(1 – 40)ox and Aβ(1 – 42)ox.(16) The sum of the signal
volumes with chemical shifts between 0.0 and 1.5 ppm in the 1D 1HNMRspectra of
freshly prepared aqueous solutions of Aβ(1 – 40)ox and Aβ(1 – 42)ox is plotted vs. the
time that has elapsed after the sample preparation. (Reproduced from R. Riek,
P.Gu¨ntert, H. Do¨beli, B.Wipf, K. Wu¨ thrich,‘NMR Studies in Aqueous Solution Fail to
Identify Significant Conformational Differences between the Monomeric Forms of two
Alzheimer Peptides with Widely Different Plaque-Competence, A β(1–40)ox and A β(1–
42)ox’, Eur. J. Biochem., 268, 5930–5936 (2001).© Blackwell Publishing Ltd, 2001.)

36

Solution state NMR is the most useful tool when monomer of
proteins and amyloidogenic peptide has to be detected. However,
when detections are to be carried out for higher oligomeric states
thee signals tend to be broad and eventually give negligible signals or
disappear due to an increase in the tumbling time. Further, the basic
nature of amyloid proteins being the heterogenic and use of high
concentrations required for carrying out the experiments pose more
difficulties in studies of oligomer rich solution state NMR
spectroscopy.

37

III. X-Ray Fiber Diffraction

Figure 7: Electron microscope image of amyloid fibrils by negative
staining of IAPP
(a) Electron microscope image of amyloid fibrils formed by negative staining using IAPP.
Fibrils are long, unbranched and approximately 100nm in diameter. b) X-Ray diffraction
pattern showing the position of 4.7Å meridonial and 10Å equatorial reflection in a
cross-β pattern (Reproduced from O.S. Makin, L.C. Serpell, ‘Structures forAmyloid
Fibrils’, FEBS J., 272, 5950–5961 (2005). ©Blackwell Publishing Ltd.)
38

X-Ray Fiber diffraction method utilized low quality and
pulverized crystalline samples and provides information about the
orientation of the molecules in the test sample. Amyloid fibers
exhibit broad diffraction patterns of equatorial reflections at 10Å and
meridonial reflections are 5Å
18
. Equatorial reflections provide a
measurement of the corresponding space between the
protofilaments while the meridonial reflections are an estimate of
the number of protofilaments in the fibril.

39

IV. Dynamic Light Scattering Spectroscopy
Dynamic Light Scattering spectroscopy, commonly referred to
as DLS is a widely used, non-invasive and non-destructive method
used for quantitative measurements by manipulating the optical
properties of the aggregates and determining their diffusion
coefficient when the aggregates are in constant Brownian motion in
the solution or suspension. The calculations are based on the Stokes-
Einstein equation
19
:

Where,
D= diffusion coefficient
K
b
= Boltzmann’s constant
T= Absolute temperature
40

η = Viscosity of suspending liquid
R
h
= Hydrodynamic radius
D
h
= Hydrodynamic diameter
DLS is the most well suited analytical technique if the
measurement of large size of aggregates is of primary concern. This is
because of the fact that the intensity of light scattering is directly
proportional to the square of the particle mass. Furthermore, the
angle of scattering of light is in variation with varied shapes of
aggregates like rod-shaped aggregates or unfolded structures. If the
shape of the protein is known then it is very easy to resolve the
monomeric and dimeric state of the protein, however it is not able to
differentiate between oligomers if their hydrodynamic radius has a
difference by the factor of 2.Use of DLS is widespread for structural
studies of amyloidogenic proteins like barstar, calcitonin, insulin etc.
There are various other analytical techniques that are also widely
used in detection of aggregates and are suitable for quantifying the
extent of aggregation.
41

CHAPTER 5
INHIBITION OF PROTEIN AGGREGATION

The most important and widely discussed topic throughout the
biopharmaceutical industry is the techniques employed on reducing the
most common problem of aggregation and developing ways in order to
inhibit the aggregates on developing while the protein formulation is
processed in the production batch.
I. Common Strategies
1

Approaches to inhibition of protein aggregation can be divided into
four general approaches:
1. Internal modification of protein structure
2. External modification (environmental factors) of the protein
structure
3. Site directed mutagenesis
4. Chemical reactions
Amongst all such approaches, it is most important to keep in mind that
introducing structural and inherent modifications in the protein for
42

effective stabilization, there should be little effect on the biological
activity of the protein. Although it is not possible to retain the same
biological activity, every possible strategy should be employed in order
to maintain the risk-benefit ratio. Illustrations of past experiments on
developing such a strategy showed desirable results. For e.g. Insulin
when conjugated with methoxypoly ethylene glycol showed increased
resistance to shaking-induced fibrillation
1
. Various additives or
excipients are used to manipulate the protein environment and lead to
inhibition of protein aggregation. Common examples include salts,
sugars, polyols, polymers, surfactants etc. These additives mainly work
by increasing the stability of the protein and preventing surface-charge
interactions that might lead to aggregation. Other approaches also
include increasing solvent viscosity, increased rigidity and reduction of
solvent accessibility and conformational mobility. The following
approaches discuss in detail the various strategies used for inhibiting
protein aggregation.

43

Table 3: Approaches towards inhibition of protein aggregation
Ref: Wei Wang. Protein aggregation and its inhibition in biopharmaceutics; International Journal
of Pharmaceutics 289 (2005) 1-30

44

II. Specific Strategies:
Approaches for inhibition of protein aggregation:
1. Temperature
During protein folding a certain temperature is required in
order to maintain an optimum thermal motion between the
molecules so that they cross the energy barrier and reach the folding
transition state. However, increase in the temperature of the protein
solution causes an increase in both inter-molecular interaction forces
and hydrophobic interactions ultimately increasing the chances of
protein aggregation.
Studies carried out by Klein and Dhurjati in 1995 showed that
at temperatures higher than 35
0
C the higher rate of folding is
associated with an equal increase in protein aggregation
20
. However,
evaluating on a broader scale, the rate of protein aggregation with
respect to temperature is purely dependent on the protein itself. For
example, in the experiments carried out by Xie and Wetlaufer in 1996
45

on bovine CAD showed an increased refolding yield at 20
0
C
temperature compared to subsequent temperatures of 4
0
C and
36
0
C
21
.
2. Freezing
Freezing is the most common and widely used method for
storage of proteins over a long period of time. However, protein
freezing also is one of the causes of inducing protein aggregation and
has to well understand before subjecting the newly produced protein
to extremely low and harsh temperatures. Freezing induces one of
the following stresses on the proteins
1
:
a. Formation of ice-water interfaces:
b. pH change:
c. Low temperature
d. Solute concentration change
e. Phase separation
Sudden changes in temperature of the protein solution cause
undesirable changes and affect its stability. Proteins that denatured
46

due to formation of an ice-water interface must be introduced to
lower temperatures at a very slow and steady rate. Sudden and
extreme changes in temperature may cause the protein to build up
between such interface and damage protein the integrity. On the
other hand, slower rate of lowering the temperature increases the
possibility of crystal formation or initiation of crystallization that can
serve as a nucleation or seeding mechanism and induce protein
aggregation
1
.
Protein solutions are composed of pure protein molecules
along with storage buffers and other excipients needed to maintain
the viability of the protein. However, changes in temperature may
induce pH changes in the buffer solutions and become a frequent
cause of resulting protein aggregation. Hence, suitable buffers must
be used in order to prevent the initiation of aggregation.
Other factors like increase in solution viscosity, stearic
hindrance and increased protein-protein interactions can also serve
as potential reasons towards protein aggregation. Most commonly,
47

surfactants like Tween 80 and Tween 20 are utilized to effectively
manage pH and decrease surface interactions during the freezing-
thawing cycle.
3. Drying
Proteins in the normal hydrated state are generally covered by
a certain amount of water (0.3g/g of protein) around it in order to
maintain its native state
22
. Processes of drying usually removes this
hydrated layer and may render the protein susceptible to changes in
conformation and shape disruption ultimately leading to aggregation.
Hence, care should be taken in order to maintain the protein in its
hydrated state and that the process of drying should not affect the
immediate surrounding hydrated layer. For such instances, excipients
are used which form a hydrogen bond with the protein replacing the
need of polar bonding requirements on the protein surface. Such
excipients can also be termed as water substitutes and are generally
amorphous in nature. Studies show that such an amorphous glass
formation is a prerequisite during dehydration processes of the
48

protein. Examples of commonly used excipients are sugars like
trehalose and sucrose and polymers like hydroxyethyl cellulose
during lyophilization.
4. Protein concentration
Increase in protein concentration gives more incidences of
possible protein-protein interaction and hence increased probability
of protein aggregation. Studies carried out that simulate in-vitro
protein folding depict that higher concentrations of protein causes a
major fold increase in protein aggregation than it does in protein
folding rate. Moreover, the sizes of aggregate formed are also
comparatively larger. Hence, low concentrations are favored during
the protein refolding processes. Optimum concentrations for
refolding are desirable to be less than 1mg/ml
23
.However, at very low
concentrations there is a possibility of loss or denaturation of the
protein due to surface adsorption and hence while conducting
refolding experiments for unknown proteins, the best recommended
range is generally narrow and falls between 10 to 50µg/ml
24
.
49

5. Denaturant concentration
1

Denaturant agents are generally used in the protein solution to
maintain protein solubility. However, its concentration in the
solution strongly affects the rate and extent of protein refolding.
Invariably low concentrations of denaturants may not produce the
desired effect of rendering solubility properties and convert the
proteins into aggregation prone, denatured species. Higher
concentrations of denaturants cause a significant reduction of
protein-water interactions. Basically, lower concentrations of
denaturants pose a major risk towards aggregation and should be
maintained carefully. Studies show that the optimum concentration
of denaturants in protein solution should be 3.5M urea for rpGH and
higher or lower than 4.5M GdnHCl for rhGH
1
.
6. Use of additives
Molecular chaperones have shown to provide promising
results in escaping protein aggregation. This idea was proposed
based on the fact that bacteria were initially used as molecular
50

chaperones for cellular cascade pathways. Such chaperones are
believed to suppress the protein aggregation by alternate binding
and release of the resulting folded intermediates. Such an approach
being a highly sophisticated one, it requires high capital investment,
production costs and burden of introducing even more extra
purification methods. Hence, use of simpler additives shows much
more acceptability in the pharmaceutical industry where cost of
production is one of the essential feature of the company’s
objectives.Such non-protein additives are called as “artificial
chaperones”. Majority of additives used for inhibiting protein
aggregation involves use of detergents and cyclodextrins. Detergents
show their mechanism of action in suppressing the formation or
aggregates rather than just aid in solubilizing the aggregates. In a
similar way, formation of protein aggregates was prevented when
cyclodextrins were used during the renaturation of carbonic
anhydrase. Increased efficiency can be obtained by using a
51

combination of both detergents and cyclodextrins in the solution.
Other examples of additives used can be enlisted as below:
Surfactants for rhGH, MHC-II and CAB
Sucrose for β-lactamsase
PEG for rhDNase and rhtPA
Na
2
SO
4
for CAB
The mechanism of action of such additives can be explained by
their role in the stabilization of the native state, solubilizing folded
intermediates or destabilizing the incorrectly folded intermediates.
In circumstances of oxidative refolding of reduced proteins, an
optimum concentration of redox conditions is required such that
disulfide bond formation is favored. A common redox pair that
provides such conditions is the GSH/GSSG pair. Studies carried out on
oxidative renaturation of hen egg white lysosome (HENWL) required
a GSH/GSSG ratio between 0.8 and 3 in order to give best results
24
.

52

7. Shaking and shearing
Shaking results in formation of very large and exposed air-
water interface. Such an interface is a precursor to more
hydrophobic interactions of protein surface and the air phase
inducing rapid aggregation. Shearing also exposes the same
hydrophobic interactive surfaces resulting in easy aggregation once
the proteins are induced to such physical stress. As a measure to
avoid such interactions, surfactants are employed which tend to
reduce the exposure of proteins to the hydrophobic surfaces by
directly and competitively binding to the proteins. Some polymers
are also employed which increase the viscosity of the solution and
reduce the motion of the protein backbone and inhibit protein
aggregation.

53

CHAPTER 6
STORAGE OF PROTEINS
Storage of proteins
Proteins are stored for a varied interval of time in research centers.
An 18-month shelf life is the normal accepted requirement for storage of a
biopharmaceutical product. Storing protein solutions at low temperatures
up to the range of -80
0
C to -196
0
C is also the normally accepted criterion;
however, care should be taken for meeting the requirements of each
individual protein as extreme low temperatures are in itself the predictors
of protein aggregation.
The first step towards a successful approach of storing proteins for
long intervals of time is to use good buffering agents and optimum
concentrations. Further, while using liquid proteins, use of a suitable
stabilizer is very essential in order to maintain the integrity of the shape
and structure of proteins. For example, sugars and salts like NaCl which play
an important role in stabilization of proteins like α
1
- trypsin. Studies reveal
54

that use of PEG and zinc chloride for storage of α
1
-trypsin inhibits aggregate
formation at a temperature of 4
0
C.
Further, solid proteins are required to be stabilized by amorphous
excipients that form a layer around the protein molecules preventing them
from dehydration and damage to interface. As an extra controlling factor,
the moisture of the protein molecules is of very important consideration.
Increase in the moisture content above optimum levels may be a cause of
initiating aggregation. Studies carried out by Schwendeman et al., in 1995
showed that under 36% of water content during storage at 37
0
C the
lyophilized tetanus toxoid showed maximum extent of aggregation (78%)
while at temperatures below or above that level showed significantly lower
aggregation rates
25
.

55

CHAPTER 7
TOXICITIES/DISEASES: A RESULT OF PROTEIN AGGREGATION
Protein aggregation within the body is normally controlled by
complex cellular quality control mechanisms. However, under some
unusual circumstances a subset of proteins undergo aggregation within the
cytoplasmic matrix of the tissues. It is now very well evident that such an
aggregation occurs not only in the extracellular matrix but also in the
cytoplasm and the nucleus of the cells. The resulting protein deposits are
coined as amyloids which are characterized by a highly ordered, insoluble
and similar aggregate structure called the cross-β spine. An increasing
number of diseases are being identified which show their pathogenesis in
development of amyloids and protein aggregates amongst the cellular
matrix. Some of these diseases are Alzheimer’s disease, Huntington’s
disease, spinocerebellar ataxias, Parkinson’s disease, prion diseases and
more. It has now come into light that there exists a dynamic equilibrium
between the monomeric, oligomeric and higher oligomeric states of the
amyloids and that its occurrence depends on a variety of factors including
56

concentration of protein in the bulk solution and interaction with other
monomers under specific cellular environments. Hence, we need to target
the mechanism of this equilibrium achieved between the monomeric and
aggregate states in order to develop effective strategies against the
formation of such aggregates and consecutively the treatment of such
diseases.
Diseases that find their origin in protein aggregation show that they
mainly have their effect on the nervous system. In fact proteins that are
expressed ubiquitously have shown to aggregate in a major proportion in
the central nervous system (CNS). However, diseases caused by protein
aggregation are not just limited to the central nervous system. In general,
the diseases caused by aggregation of proteins are called amyloidoses. Such
diseases occur systemically within any part of the body or are restricted to
a single organ and show its deleterious effect there. For example,
aggregation and accumulation of immunoglobulin light chain amyloid fibrils
leads to AL amyloidosis wherein the deposits may accumulate in almost any
part of the body. Serum amyloid A protein and its catabolic products also
57

tend to accumulate systemically and are termed as Reactive systemic
amyloidosis. Further, accumulation of transthyrettin (TTR) leads to
development of Amyloid Transthyretin (ATTR). Presence of amyloidosis
which is restricted to a single organ can be illustrated in type 2 diabetes
wherein there is accumulation of islet amyloid polypeptide in the pancreas’
cellular matrix.
Our discussion here will be majorly focused on two diseases:
Alzheimer’s disease and prion disease.
I. Alzheimer’s disease
26

Alzheimer’s disease is one of the most widespread
neurodegenerative disorders leading to dementia and hence receiving the
highest amount of focus for developing a successful strategy for effective
combat of the disease. This disease was first coined by a German
psychiatrist Alois Alzheimer in 1906 A.D. Alzheimer is generally called the
disease of the aged as most of diagnosis occurs in people aged 65 or above.
The disease progresses once diagnosed and due to no effective and
promising cure it eventually leads to the death of the individual. Symptoms
58

of this disease are sometimes confused with age-related problems that
people face in their day to day life. Initial symptoms recorded are difficulty
in remembering recent events of life or even routine activities like eating,
reading, going to nearby neighborhood places, remembering names of
people etc. Eventually as the disease progresses it may also incorporate
disturbed thinking ability of the patient, confusion, irritability, mood
swings, stress, aggressive behavior, trouble with language and ability to
remember names and places. Gradually the body functions lose their
optimum capability rendering the patient incapable of living a normal life.
The exact cause and mechanism of this disease is still under study and a
variety of approaches are put forward targeting different proposed
mechanisms ultimately with the hope of completely mitigating this disease
and helping the patient live a normal life again. Current treatments show an
improvement in the field of managing symptoms of the disease and up to
some extent managing the amount of plaque formation and decreasing the
damage caused to the brain.
59

This diseases’ pathology is characterized mainly by the formation of
protein aggregates in the brain. Two kinds of aggregates are formed:
Amyloid plaques and intracellular neurofibrillary tangles
26
. Figure 8a depicts
an amyloid-β (Aβ)-specific antibody stained section of the cortical section of
human brain of a patient suffering from Alzheimer’s disease. Presence of
extracellular lesions called the amyloid or senile plaques are a hallmark of
the presence of Alzheimer’s disease and are predominantly composed of
Aβ peptide. Figure 8b depicts a phosphor-tau specific antibody stained
section of human cortical brain. These lesions are developed as a result of
neurofibrillary tangles and are a second hallmark of the intraneuronal
lesions of the presence of Alzheimer’s' disease. Intraneuronal lesions are
predominantly composed of hyperphosphorylated and abnormal filaments
of microtubule associated tau protein.
60

Figure 8: Stained section of Human cortical brain of Alzheimer’s
patient a) Human cortical section of Alzheimer’s patient stained with amyloid Aβ
specific antibody. b) Human cortical section of Alzheimer’s patient stained with
phosphor-tau-specific antibody
(Adriano Aguzzi and Tracy O’Connor; Protein aggregation diseases: pathogenecity and
therapeutic perspectives. Nature Reviews 9, 237–248 (March 2010))

61

Imbalances in Aβ homeostasis are characterized at a very early stage in
development of Alzheimer’s disease and this is proved by genetic
evidence
27
. Figure 9 depicts a ribbon diagram of a three dimensional
structure of amyloid-β (Aβ42) (residues 17 to 40). On aggregation, amyloid
proteins are thought to produce a same kind of three dimensional
structures which can be termed as the cross-β spine or amyloid. Cross-β
spine is characterized by an orderly arrangement of thick β-sheets.

62

Figure 9: Three-dimensional ribbon diagram of Aβ-
amyloid protein in cross β-spine conformation
Ribbon diagram of amyloid Aβ-amyloid three dimensional
structures formed in a cross β-spine.
(Adriano Aguzzi and Tracy O’Connor; Protein aggregation diseases:
pathogenecity and therapeutic perspectives. Nature Reviews
9, 237–248 (March 2010))

63

Studies carried out in determining the mechanism of formation of such
amyloids reveals the following molecular cascade and is widely accepted
(Figure 10).

Figure 10: Proposed mechanism of Amyloid-β formation
(Adriano Aguzzi and Tracy O’Connor; Protein aggregation diseases:
pathogenecity and therapeutic perspectives. Nature Reviews 9, 237–248
(March 2010))

64

Investigational
approach
Name of treatment Expected outcome Stage of approval
Inhibit the formation
of amyloid in the
cellular matrix
γ – secretase
inhibitors and
modulators
Formation of Aβ40
or Aβ42 by reduction
in carboxy terminal
cleavage of APP
In Phase II and
Phase III clinical
trials

β - secretase inhibitors Formation of Aβ40
or Aβ42 by reduction
in amino terminal
cleavage of APP
In Phase I clinical
trials
Promotion of
amyloid clearance
Aβ immunotherapy Increased clearance
of Aβ containing
aggregates
Phase II and III
clinical trials

Prion immunotherapy Enhanced clearance
of PrPSc-containing
aggregates;
prevention of the
invasion
of prions into
neurons
Preclinical
Inhibition of amyloid
aggregation
Scyllo-inositol Prevention of the
formation of
higher-order
aggregates
Phase II clinical trials
Tafamidis Stabilization of the
native state of
Transthyretin
Phase II and III
clinical trials
Table 4: Summary of therapeutic strategies for protein aggregation
diseases
(Abstracted from Adriano Aguzzi and Tracy O’Connor; Protein aggregation diseases:
pathogenecity and therapeutic perspectives. Nature Reviews 9, 237–248 (March 2010))

65

The above mentioned treatment strategies make an important assumption
that the formation of amyloid plaques are toxic and the only reason for
development of the disease. Hence, all the strategies aim at either reducing
the amyloid formation or increase in its clearance. However, recent studies
show that formations of plaques have very little in correlation to the
degree of underlying dementia
28
. In fact, more recent and sophisticated
observations have revealed that the total load of Aβ aggregates is the true
predictor of the cognitive performance of the individual. Scientists have
come up with theories for explaining the above mentioned hypothesis, that
the upon histological examination of brains of healthy and Alzheimer’s
patients, it can be observed that amyloidal aggregates in a healthy
individual with a fair level of cognitive function show a stable and begnin
position in their site of appearance whereas in case of Alzheimer’s' patients
the amyloidal aggregates are in a kinetic state; in a constant equilibrium
between the monomer and oligomeric state
29, 30
. Such aggregates exist in
the form of monomers, dimers, trimmers and higher oligomeric
intermediate forms and are highly toxic, insoluble aggregates
31, 32
. This
66

observation received even more support when studies carried out on APP-
transgenic mice receiving scyllo-ionsitol (ELND005) treatment showed a fair
reduction in the total load of Aβ aggregates but not Aβ trimmers and co-
incidentally showed an improvement in the cognitive function
33
. This
theory put forward opens up a totally new outlook into the therapeutic
strategies that are traditionally used to treat Alzheimer’s disease. Indeed,
the studies carried out APP-transgenic mice put forward the results that
there is significant improvement in cognitive function even without the
clearance of Aβ aggregates from the body. Strategies that target such
conformation specific aggregates, identify and neutralize the toxic species
of oligomers in combination with increased clearance of Aβ aggregates
from the brain can be utilized in order to devise a more effective treatment
regimen towards improving the cognitive function in Alzheimer’s disease.

67

II. Prions Disease
26

A very important feature of this disease is that this disease is
transmissible across species; humans and across. Hence, it is also known as
transmissible spongiform encephalopathies that include Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia and Creutzfeldt-
Jakob disease in humans. Across species they are identified under various
names like: bovine spongiform encephalopathy in cattle, chronic wasting
disease in cervids and scrapie in sheep
34
.
Once infected by this disease, its incubation period being very long it
becomes impossible to detect and start the treatment for this disease until
the disease has reached its full blown state and started showing symptoms.
Once symptoms start to appear the progression of this disease occurs at an
exponential level ultimately leading to the death of an individual. Some
commonly observed symptoms include dementia, convulsions, difficulty in
maintaining balance and coordination of the body and personality along
with behavioral changes
26, 34
. Transmission of prion disease is believed to be
by acquired, familial or sporadic way. Assumptions are made that upon
68

interaction of the normal prion protein with the toxic prion protein
conformation, there follows a series of steps which makes the remaining
surrounding healthy prion proteins to convert into the toxic conformations.
In normal conditions, the prion protein is present in its native conformation
(PrP
c
); however, in case of a diseased condition this protein has an altered
conformation (PrP
Sc
) which undergoes accumulation and deposits on the
neurons
26
. Figure 4a depicts a histological section of the brain cortex of a
patient affected by prions disease. Presence of insoluble deposits revealed
by prion protein (Pr-P) specific antibody staining (Fig 11a) depicts the
accumulation of PrP aggregates folded in an abnormal conformation (PrP
Sc
).
Another cortical section (Fig 11b) stained with haematoxylin and eosin
shows the presence of large vacuoles, also termed as spongiosis. Such
lesions are believed to be the key factors responsible for the transmission
ability of spongiform encephalopathies.

69

Figure 11: Stained sections of human cortical brain of Prions’
disease diagnosed patient A) Human brain cortical section stained with PrP
specific antibodies showing presence of altered conformation PrP
Sc
aggregates. B)
Human brain cortical section stained with haematoxylin and eosin showing presence
of vacuoles and lesions believed to be responsible for the transmission ability of the
disease. C) Proteolytic degradation followed by PrP
Sc
-specific immunostaining (PrP
Sc
-
histoblot) of coronol mouse brain sections from C57BL/6 (prion susceptible) and
Prnp
-/-
(prion resistant) mice infected with a mouse-adapted prion strain, Rocky
Mountain Laboratory passage 5 (RML5)
(Heikenwalder, M.et al. Germinal center B cells are dispensable in prion transport
and neuroinvasion. J. Neuroimmunol. 192, 113–123 (2007))

70

PrP
Sc
aggregates are altered conformational structures of the PrP
c

native protein. The conversion of the native conformation to PrP
Sc
involves
a large number of cellular co-factors. However, the end product, PrP
Sc

aggregates are the sole infectious agents. The conformation change process
is highly detectable and involves the dramatic increase in presence of PrP
Sc

compositions upon association with the PrP
Sc
conformers. The dramatic
shift from native conformation to a secondary structure is detected by
Figure 12: SDS-PAGE of PrP-specific brain homogenates digested
by proteinase K: A PrP-specific immunoblot of brain homogenates digested by
proteinase K (PK) and separated by SDS–PAGE, taken from RML5-infected C57BL/6
(PrPSc) or uninfected (PrPC) mice. PrPSc and PrPC have identical amino-acid
sequences and are thought to differ only in secondary structure. Although the crystal
structure of PrPSc has not been solved, its conformation can be distinguished from
that of PrPC using biochemical techniques.
(Heikenwalder, M.et al. Germinal center B cells are dispensable in prion transport and
neuroinvasion. J. Neuroimmunol. 192, 113–123 (2007))

71

initial protein composition of approximately 45% α helices and few β-sheets
to 30% α-helices and approximately 45% β-sheets (Fig 11c, 12). Such a
conformational change is accompanied with change in physicochemical
properties including the resistance to degradation and proteinase K
digestion. Under normal circumstances, the prion protein in its native
conformation is present on the cell surface and is a permanent resident of
the lipid rafts. (Figure: 13a) There seems to be a possibility that this native
conformation may serve as a binding site to potentially large amount of
extracellular toxic species and target them towards lysosomes ultimately
resulting in degradation. (Figure: 13b) Hence, under situations when there
is an accumulation of toxic extracellular species on the PrP
c
, it may induce
conformational changes causing the formation of PrP
Sc
and a direct
stimulation to initiation of cell death pathways.

72

The altered physicochemical properties serve as a key factor in
distinguishing between the native and toxic conformations as the native
protein is entirely degraded by proteinase K whereas the PrP
Sc

conformation is only partially degraded by Proteinase K digestion
26
. The
propagation of prion disease is associated with such unusual properties
that developing a straightforward and effective therapeutic strategy has
become even more difficult. However, as more studies are being conducted
Figure 13: Proposed mechanism of PrP
c
mediated oligomer
formation and initiation of toxicity: a) & b) Possible mechanism of PrP
c

mediated oligomer formation and toxicity.
(Adriano Aguzzi and Tracy O’Connor; Protein aggregation diseases: pathogenecity and
therapeutic perspectives. Nature Reviews 9, 237–248 (March 2010))

73

on the targeting of such aggregates there is a wide scope for new
opportunities and a broad vision to explore unique and developing
strategies.
As discussed in the scenario of Alzheimer’s and Prion disease, there
still remains a lot work to be done upon in order to overcome such
challenges of amyloid therapeutics. New hopes are created by the clinical
trials that are currently being carried out for secreatase inhibitors and anti-
aggregation compounds and other immunotherapeutics using animal
models.
74

CHAPTER 8
CONCLUSION

The widespread problem of protein aggregation in the
biopharmaceutical industry is raising concerns on developing a certain
strategy that will be 100% effective and give a hope in successful
development of a biopharmaceutical product. Strict FDA and GMP
guidelines allow the presence of aggregates in a protein formulation up to
the order of parts per million. Such high purity solutions need to be
maintained and stored for long durations of time and still are viable and
free of any impurities during the entire interval from production to
consumption. Although the protein sequence is a predictor of the inherent
aggregation abilities, a lot of other external factors play an important role
in controlling and affecting the rate and extent of protein aggregation. In
spite of a wide variety of analytical techniques available for detecting and
preventing aggregation processes the final decision remains in the hands of
the experienced researcher on when and how to utilize these techniques.
75

Variations in their accuracy, precision, ease of accessibility, quantification
abilities are the parameters that the researcher needs to evaluate and
hence decide the best possible approach towards protecting his protein
solution from aggregation. Recognizing the causes and mechanisms of
protein aggregation is the first and foremost essential feature of preventing
the protein from aggregation.
In spite of such a wide variety of analytical techniques, pool of
excipients and knowledge of the external factors responsible for
aggregation this topic has still remained as a major issue of discussion. Trial
and error kind of approaches have become a main stay in the effective
combat against protein aggregation partly due to our lack of understanding
of manipulating each and every possible step during the biopharmaceutical
product formulation that have the slightest risk developing aggregation.
Hence, a very thorough and detailed investigation, probably for detecting
that particular key points during the production and storing process that
pose aggregation risks must be carried out. Further, use of a combination of
the above mentioned techniques for inhibiting protein aggregation must be
76

utilized for a more effective control towards any chances or possible
aggregation. Lastly, after such a large amount of investment being made in
developing a high quality and extra-pure biopharmaceutical formulation,
care should be taken for meeting the optimum storage conditions and
hence developing a hopeful ultimate cure of all problems of protein
aggregation in the biopharmaceutical industry.

77

REFERENCES

1. Wei Wang, “Protein aggregation and its inhibition in
biopharmaceutics”. International Journal of Pharmaceutics, 2000.
289: p. 1-30

2. Christopher A Ross & Michelle A Poirier, “Protein aggregation ad
neurodegenerative disease”. Nature Medicine Reviews:
Neurodegeneration, July 2004. p. 510-518

3. Liu, Jun et al., “Reversible Self Association Increases the Viscosity of a
Concentrated Monoclonal Antibody in Aqueous Solution”. Journal of
Pharmaceutical Sciences, 2005. 94(5): p. 1928-1940

4. J. Timothy Westwood and Carl Wu. “Activation of Drosophila heat
Shock Factor: Conformational Change Associated with a Monomer-
to-Trimer Transition”. Molecular and Cellular Biology, 1993. 13(6): p.
3481-3486

5. Karasawa, Akira et al., “Detection of Oligomerization and
Conformational Changes in the Na
+
/H
+
Antiporter from helicobacter
pylori by Fluorescence Resonance Energy transfer”. The Journal of
Biological Chemistry, 2005. 280(51): p. 41900-41911

6. Kazuhiko Adachi and Toshio Asakura, “Nucleation-controlled
Aggregation of Deoxyhemoglobin S.” The Journal of Biological
Chemistry, 1979. 254(16): p. 7765-7771

7. Hakan Nygren and Manne Stenberg, “Surface-induced aggregation of
ferritin: Kinetics of adsorption to a hydrophobic surface”. Biophysical
Chemistry, 1990. 38(1-2): p. 67-75

8. Linda O. Narhi et al., “Classification of Protein Aggregates”. Journal of
Pharmaceutical Sciences, 2012. 101(2): p. 493-498
78

9. Goda S, Takano K, Yamagata Y, Nagata R, Akutsu H, Maki S, Namba K,
Yutani K. Protein Science 2000. 9: p. 369–375.

10. Tadashi Ueda et al., “Aggregation and chemical reaction in Hen
Lysosome caused by heating at pH 6 are depressed by osmolytes,
sucrose and trehalose”. Journal of Biochemistry, 2001. 130(4): p.
491-496

11. Harald Forbert et al., “Aggregation-Induced Chemical Reactions: Acid
Dissociation in Growing Water Clusters.” Journal of American
Chemical Society, 2011. 131(11): p. 4062-4072

12. Eva Y. Chi et al., “Physical Stability of Proteins in Aqueous Solution:
Mechanism and Driving Forces in Nonnative Protein Aggregation”.
Pharmaceutical Research, 2003. 20(9): p. 1325-1336

13. Vic´ens M. “Pharmaceutical Formulation & Quality”.
December/January 2009.

14. Huiyuan Li, Farid Rahini, Sharmishtha Sinha, Pancham Maiti and Gal
Bitan, Kazuma Murakami. “Amyloids and Protein Aggregation-
Analytical Methods”. Encyclopedia of Analytical Chemistry R.A
Meyers (Ed.), 2009. p. 1-32

15. Y. Fezouie, D.B. Teplow. “Kinetic studies of amyloid-β protein fibril
assembly. Differential Effects of α-helix stabilization”. Journal of
Biological Chemistry, 2002. 277: p. 36948-36954

16. Vrkljan, M Foster, T.M., et al.,”Thermal stability of low molecular
weight urokinase during heat treatment.II Effect pf polymeric
additives”. Pharm. Res. 1994. 11: p. 1004-1008

79

17. Kwon, Y. M., Baudys, M. et al., “In situ study of insulin aggregation
induced by water-organic solvent interface”. Pharm. Res., 18: p.
1754-1759

18. M. Sunde, et al., “Common core structure of amyloid fibrils by
synchrotron X-Ray Diffraction”. Journal of Molecular Biology, 1997.
273: p. 729-739

19. A. Einstein, “On the Motion of Small particles suspended in liquids at
rest required by the molecular-kinetic theory of heat”. Ann. Phys.,
1905. 17: p. 549-560

20. Klein, J., Dhurjati, P., “Protein aggregation kinetics in an Escherchia
coli strain overexpressing a Salmonella typhimurium, CheY mutant
gene”.Appl. Environ. Microbiol., 1995. 61: p. 1220-1225

21. Xie, Y., Wetlaufer, D.B., “Control of aggregation in protein refolding:
the temperature-leap tactic”. Protein Science, 1996. 5: p.517-523

22. Rupley, J.A and Careri, G., “Protein hydration and function”.
Advances in Protein Chemistry, 1991. 41: p. 37-172

23. Cleland, J. L., Wang, D. I., “Refolding and aggregation of bovine
carbonic anhydrase B: quasi-elastic light scattering analysis”.
Biochemistry, 1990. 29: p. 11072-11078

24. Hevehan, D., De bernardez-Clark, E., “Oxidative renaturation of
lysosome at high concentrations.” Biotechnology & Bioengineering,
1997. 54: p. 221-230

25. Schwendeman, S. P., Costantino, H. R. et al., “Stabilization of tetanus
and diphtheria toxoids against moisture-induced aggregation”. Proc.
Natl. Acad. Sci. US. 1995. 92: p. 11234-11238

80

26. Adriano Aguzzi and Tracy O’Connor, “Protein aggregation diseases:
pathogenecity and Therapeutic perspectives”. Nature Reviews: Drug
Discovery, 2010.l 9: p. 237-248

27. Masters, C. L. et al., “Amyloid plaque core protein in Alzheimer
disease and Down syndrome”. Proc. Natl. Acad. Sci. USA 1985. 82:p.
4245-4249

28. Wilcock, G. K & Esiri, M. M. “Plaques, tangles and dementia. A
quantitative study.” Journal of Neurological Sciences, 1982. 56: p.
343-356

29. Podlisny, M. B et al., “Aggregation of secreted amyloid β-protein into
sodium dodecyl sulfate-stable oligomers in cell culture.” Journal of
Biological Chemistry, 1995. 270:p. 9564-9570

30. Walsh, D. M., Tseng, B. P., Rydel R. E., Podlisny, M. B & Selkoe, D. J.
“The oligomerization of amyliod β-protein begins intracellularly in
cells derived from human brain”. Biochemistry, 2000. 39: p. 10831-
10839

31. Gong, Y. et al., “Alzheimer’s disease-affected brain: presence of
oligomeric Aβ ligands (ADDLs) suggests a molecular basis for
reversible memory loss”. Proc. Natl. Acad. Sci. USA. 2003. 100:p.
10417-10422

32. Lesne, S. et al., “A specific amyloid-β protein assembly in the brain
impairs memory.” Nature, 2006. 440: p. 352-357

81

33. McLaurin, J. et al., “Cyclohexanehexol inhibitors of Aβ aggregation
prevent and reverse Alzheimer phenotype in a mouse model.”
Nature Med. 2006. 12: p. 801-808

34. Aguzzi, A., Sigurdson, C & Heikenwaelder, M., “Molecular
mechanisms of prion pathogenesis.” Annual Review on Pathology.
2008. 3: p. 11-40

82

COMPREHENSIVE REFERENCES
Adriano Aguzzi and Tracy O’Connor, “Protein aggregation diseases:
pathogenecity and Therapeutic perspectives”. Nature Reviews: Drug
Discovery, 2010.l 9: p. 237-248

Aguzzi A., Sigurdson, C & Heikenwaelder, M., “Molecular mechanisms of
prion pathogenesis.” Annual Review on Pathology. 2008. 3: p. 11-40

Christopher A. Ross & Michelle A Poirier,“Protein aggregation ad
neurodegenerative disease”. Nature Medicine Reviews:
Neurodegeneration, July 2004. p. 510-518

Cleland, J. L., Wang, D. I., “Refolding and aggregation of bovine carbonic
anhydrase B: quasi-elastic light scattering analysis”. Biochemistry, 1990.
29: p. 11072-11078

Einstein A., “On the Motion of Small particles suspended in liquids at
rest required by the molecular-kinetic theory of heat”. Ann. Phys., 1905.
17: p. 549-560

Eva Y. Chi et al., “Physical Stability of Proteins in Aqueous Solution:
Mechanism and Driving Forces in Nonnative Protein Aggregation”.
Pharmaceutical Research, 2003. 20(9): p. 1325-1336

Fezouie Y., D.B. Teplow. “Kinetic studies of amyloid-β protein fibril
assembly. Differential Effects of α-helix stabilization”. Journal of
Biological Chemistry, 2002. 277: p. 36948-36954

Goda S, Takano K, Yamagata Y, Nagata R, Akutsu H, Maki S, Namba K,
Yutani K. Protein Science 2000. 9: p. 369–375.

83

Gong, Y. et al., “Alzheimer’s disease-affected brain: presence of
oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible
memory loss”. Proc. Natl. Acad. Sci. USA. 2003. 100:p. 10417-10422

Hakan Nygren and Manne Stenberg, “Surface-induced aggregation of
ferritin: Kinetics of adsorption to a hydrophobic surface”. Biophysical
Chemistry, 1990. 38(1-2): p. 67-75

Harald Forbert et al., “Aggregation-Induced Chemical Reactions: Acid
Dissociation in Growing Water Clusters.” Journal of American Chemical
Society, 2011. 131(11): p. 4062-4072

Hevehan, D., De bernardez-Clark, E., “Oxidative renaturation of
lysosome at high concentrations.” Biotechnology & Bioengineering,
1997. 54: p. 221-230

Huiyuan Li, Farid Rahini, Sharmishtha Sinha, Pancham Maiti and Gal
Bitan, Kazuma Murakami. “Amyloids and Protein Aggregation-Analytical
Methods”. Encyclopedia of Analytical Chemistry R.A Meyers (Ed.), 2009.
p. 1-32

Karasawa, Akira et al., “Detection of Oligomerization and
Conformational Changes in the Na
+
/H
+
Antiporter from helicobacter
pylori by Fluorescence Resonance Energy transfer”. The Journal of
Biological Chemistry, 2005. 280(51): p. 41900-41911

Kazuhiko Adachi and Toshio Asakura, “Nucleation-controlled
Aggregation of Deoxyhemoglobin S.” The Journal of Biological
Chemistry, 1979. 254(16): p. 7765-7771

Klein, J., Dhurjati, P., “Protein aggregation kinetics in an Escherchia coli
strain overexpressing a Salmonella typhimurium, CheY mutant
gene”.Appl. Environ. Microbiol., 1995. 61: p. 1220-1225

84

Kwon, Y. M., Baudys, M. et al., “In situ study of insulin aggregation
induced by water-organic solvent interface”. Pharm. Res., 18: p. 1754-
1759

Lesne, S. et al., “A specific amyloid-β protein assembly in the brain
impairs memory.” Nature, 2006. 440: p. 352-357

Linda O. Narhi et al., “Classification of Protein Aggregates”. Journal of
Pharmaceutical Sciences, 2012. 101(2): p. 493-498

Liu, Jun et al., “Reversible Self Association Increases the Viscosity of a
Concentrated Monoclonal Antibody in Aqueous Solution”. Journal of
Pharmaceutical Sciences, 2005. 94(5): p. 1928-1940

Masters, C. L. et al., “Amyloid plaque core protein in Alzheimer disease
and Down syndrome”. Proc. Natl. Acad. Sci. USA 1985. 82: p. 4245-4249

McLaurin, J. et al., “Cyclohexanehexol inhibitors of Aβ aggregation
prevent and reverse Alzheimer phenotype in a mouse model.” Nature
Med. 2006. 12: p. 801-808

Podlisny, M. B et al., “Aggregation of secreted amyloid β-protein into
sodium dodecyl sulfate-stable oligomers in cell culture.” Journal of
Biological Chemistry, 1995. 270:p. 9564-9570

Rupley, J.A and Careri, G., “Protein hydration and function”. Advances in
Protein Chemistry, 1991. 41: p. 37-172

Schwendeman, S. P., Costantino, H. R. et al., “Stabilization of tetanus
and diphtheria toxoids against moisture-induced aggregation”. Proc.
Natl. Acad. Sci. US. 1995. 92: p. 11234-11238

85

Sunde M., et al., “Common core structure of amyloid fibrils by
synchrotron X-Ray Diffraction”. Journal of Molecular Biology, 1997. 273:
p. 729-739

Tadashi Ueda et al., “Aggregation and chemical reaction in Hen
Lysosome caused by heating at pH 6 are depressed by osmolytes,
sucrose and trehalose”. Journal of Biochemistry, 2001. 130(4): p. 491-
496

Timothy J., Westwood, Carl Wu. “Activation of Drosophila heat Shock
Factor: Conformational Change Associated with a Monomer-to-Trimer
Transition”. Molecular and Cellular Biology, 1993. 13(6): p. 3481-3486

Vic´ens M. “Pharmaceutical Formulation & Quality”. December/January
2009.

Vrkljan, M Foster, T.M., et al.,”Thermal stability of low molecular weight
urokinase during heat treatment.II Effect pf polymeric additives”.
Pharm. Res. 1994. 11: p. 1004-1008

Walsh, D. M., Tseng, B. P., Rydel R. E., Podlisny, M. B & Selkoe, D. J. “The
oligomerization of amyliod β-protein begins intracellularly in cells
derived from human brain”. Biochemistry, 2000. 39: p. 10831-10839

Wang Wei, “Protein aggregation and its inhibition in biopharmaceutics”.
International Journal of Pharmaceutics, 2000. 289: p. 1-30

Wilcock, G. K & Esiri, M. M. “Plaques, tangles and dementia. A
quantitative study.” Journal of Neurological Sciences, 1982. 56: p. 343-
356

Xie, Y., Wetlaufer, D.B., “Control of aggregation in protein refolding: the
temperature-leap tactic”. Protein Science, 1996. 5: p. 517-523

Abstract (if available)

Abstract Aggregation of proteins is the major topic of discussion and debate in the biopharmaceutical industry. Various chemical and physical properties of proteins have been studied and manipulated in order to overcome this major hurdle during biopharmaceutical product formulation. Huge investments are done in order to understand the mechanisms underlying abnormal protein aggregation during the production process as well as in-vivo. Theories explaining this aggregation process are manipulated in the industry in order to attain the protein drug in its highest quality. Further, based on the nature and extent of such aggregates formed it is possible to classify the different types of aggregates. As aggregation is assumed to be an end-product of undesirable events occurring through the molecular cascade, based on such classification one can try to reach to the possible causative factor in the molecular cascade that leads to aggregation. Various detection techniques have been utilized to check the presence of aggregate formation while the protein drug is in its initial processing steps. Such techniques provide a lot of information on physical characteristics and real time detection on how the process of aggregation moves in its pathway. Further, we have discussed the most widely used techniques and strategies to inhibit aggregation at the in-vitro level. Storage of proteins in order to maintain it viable for a sufficiently long interval of time is very important. Techniques on effective storage and environmental conditions that may affect as a causative parameter in initiation of aggregation have been discussed. Taking a step further in this direction, many neurodegenerative disorders have found their origins in the formation of protein aggregates, called amyloids. We have discussed two major neurodegenerative diseases: Alzheimer’s disease and Prion Disease and their pathogenesis.

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Asset Metadata

Creator Shah, Manali (author)

Core Title Protein aggregation: current scenario and recent developments

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 05/16/2012

Defense Date 05/15/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aggregation,aggregation detection techniques,degenerative diseases,mechanisms of protein aggregation,OAI-PMH Harvest,protein,protein aggregates,protein aggregation diseases

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shen, Wei-Chiang (committee chair), Okamoto, Curtis Toshio (committee member), Wang, Clay C.C. (committee member)

Creator Email manalish@usc.edu,manalishah9@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-41391

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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