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Characterization of Protein Aggregates through Intrinsic

and Extrinsic Fluorescence Spectroscopy
Individual Project: Spring 2016

Dilip Rathinakumar

Of all the biological molecules found in living organisms, proteins as therapeutics
are arguably the most important biologicals in terms of clinical utility and
FOR USE]. Protein-based therapeutics can be divided into 5 groups: (a) replacing a
protein that is deficient; (b) improving an existing pathway; (c) providing a novel
function; (d) interfering with a molecule; (e) delivering other compounds. (Dimitrov).
These therapeutics are used for the therapy of cancers, immune disorders,
infections, disease, and other ailments.
Since the launch of therapeutic proteins, antibodies have become a useful tool in
the treatment of a wide range of diseases. Over time, therapeutic monoclonal
antibodies (mAb) have evolved from mice to humanized derivatives (Ratanji, 2013).
Non-human therapeutics were thought to provoke an immunogenic response due to
being a foreign derivative. Although humanized derivatives are not expected to
provoke an immune response, the formation of anti-drug antibodies (ADA) was still
noted (Sauerborn, 2010). ADA can cause adverse effects and/or reduce the efficacy
of a therapeutic protein thus immunogenicity is a key restriction for clinical use
(Ratanji, 2013).
Proteins are susceptible to aggregation and in therapeutic proteins, this can lead to
a loss of efficacy and/or provoke an immunogenic response. Methods to detect and
characterize protein aggregation are required in order to test protein formulations.
The data can be used to understand induction factors on protein aggregation as well
as improve manufacturing and storage procedures. Protein aggregates can also
influence membrane fouling during filtration and purification of proteins. Kelly
(1993) found that the bovine serum albumin (BSA) fouling of microfiltration
membranes was associated with the deposition of trace quantities of aggregated or
denatured BSA.
Common methods to characterize protein aggregates include Size-Exclusion
Chromatography, Analytical Ultracentrifugation, Asymmetrical Field Flow
Fractionation, and Dynamic Light Scattering. Of interest are fluorescence techniques
that utilize intrinsic tryptophan fluorescence or extrinsic fluorophore dyes to study
Fluorescence spectroscopy is a type of spectroscopy that analyses the fluorescence
from a sample. An excitation wavelength is chosen that corresponds to the dyes
excitation wavelength and intensity readings are taken over a range of emission
wavelength values. Fluorescence spectroscopy can be conducted with intrinsic
fluorescence due to tryptophan residues (an amino acid) present in some proteins,
or with extrinsic fluorescence through the use of fluorescent dyes that bind to
protein molecules.
Aggregation of an Immunoglobulin G antibody, induced through varying protein
concentration, ionic strength, and thermal stress, is studied using fluorescence
spectroscopy. Intrinsic Tryptophan fluorescence is compared with Nile Red Extrinsic

Literature Review
Protein Aggregation
Aggregation is often encountered during the manufacturing process including
fermentation, purification, formulation, and storage. Aggregates can cause adverse
effects that have an influence on drug efficacy as well as immune response.
Protein aggregates are defined as a universal term for all kinds of further defined
multimeric species that are formed by covalent bonds or noncovalent interactions
(Mahler, 2010). Aggregates are essentially oligomers, multimers, and non-native
assemblies as opposed to the desired species, monomers (native quaternary
structure). Figure 1 schematically shows the formation of protein aggregates from
the proteins native state to linear (ordered distribution/shape), amorphous (without
uniform shape), and visible aggregates.

Figure 1: Schematic Model of Protein Aggregation reproduced from Ratanji, 2013.

Pathways of protein aggregation differ between proteins and are affected by
environmental conditions, applied state, and the initial state of protein. In this
project, pH-induced, salt-induced, and thermal-induced aggregation are studied.
The pH of the protein solution determines electrostatic interactions due to charge
distribution on the protein surface (Mahler, 2010). In very acidic compounds, protein
cleavage occurs in which proteins breakdown into smaller polypeptides and amino

acids. The hydrolysis of peptide bonds is generally very slow but can be catalysed
(non-enzymatically) by a low pH or high temperatures. In neutral to basic solutions,
deamidation and oxidation are favoured. Deamidation is a reaction whereby an
amide functional group is removed from an organic compound. These modifications
may lead to increased aggregation. Highest levels of aggregation are generally
found at the isoelectric point (pH at which the net primary charge of a protein is
zero), as the attraction forces dominate over repulsive electrostatic forces (repulsive
forces are weak when negative and positive charges cancel each other out)
Thermal stress (increased temperature) can accelerate chemical reactions such as
deamidation and oxidation much like neutral and basic solutions. Higher
temperature has a direct effect on the proteins quaternary, tertiary, and secondary
structure thus leading to temperature-induced unfolding. (Mahler, 2010).
Fluorescence Spectroscopy
Fluorescence is the phenomenon in which an illuminating system emits light of
wavelength different from the incident light (Nahata, 2011). The incident light
excites the molecule to the highest vibrational level. Due to a phenomenon called
Internal Conversion, the molecule loses energy in a non-radiative manner to the
lowest vibrational energy level. The molecule then radiates the rest of the energy as
light of a longer wavelength and lower energy.
Fluorescence spectroscopy, or spectrofluorometry analyzes the fluorescence from a
sample using a beam of ultraviolet light that excites the samples molecules.
Fluorescence spectroscopy is similar to UV absorption spectroscopy.
Depending on the molecule, the excitation wavelength is
Fluorescent Dyes
Dye molecules are excited by the absorption of light which raises electrons from the
ground state to a higher singlet excited state. Several processes may occur which
result in energy loss: vibrational relaxation, internal conversion, solvent relaxation,
intramolecular charge transfer (ICT), and twisted intramolecular charge transfer
(TICT). When the dye reaches the lowest vibrational level, the molecules relax back
down to the ground state through fluorescence emission. (Hawe, 2007)
ICT: An electron is transferred from an electron donor group to an electron
withdrawing group in the excited dye molecules.1
TICT: A change in dye conformation such as a rotation/twist is a prerequisite for the
electron transfer to take place.
In both ICT and TICT, molecules are more likely to relax by non-radiative processes
thus leading to low fluorescence intensities of the dyes in polar environment.
(Hawe, 2007)

Nile Red
Nile Red (9-diethylamino-5-benzo[]phenoxazinone) is a lipophilic stain and
fluorescent dye that has been used for fluorescence spectroscopy (Mishra, 2011),
fluorescence microscopy of proteins (Demeule, 2007; Demeule, 2009) and lipids,
and the staining of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels
(Daban, 2001).
The fluorescence properties of Nile Red are governed by TICT with the formation of
the TICT state occurring in polar solutions while in apolar solutions, the TICT state is
thermodynamically unfavorable. (Hawe, 2007).
Nile red fluorescence is sensitive to the polarity of its environment thus can be used
to probe for changes in protein conformation related to formation of hydrophobic
surfaces. In aqueous media, it is relatively insoluble and fluorescence is strongly
quenched. Nile red fluoresces intensely when bound to a proteins hydrophobic sites
(Sackett, 1987). Aggregation and unfolding of proteins increases the amount of
hydrophobic area contributing to Nile Red binding to protein aggregates. Thus, Nile
Red can be used to study the formation of protein aggregates.
Nile Red is poorly soluble in aqueous media thus it requires organic solvents such as
water-free DMSO or ethanol to prepare stock solutions. When an organic solvent is
used, it must be ensured that it does not affect the integrity or fluorescence of the

Preparation of Nile Red Stock Solution
Nile Red is dissolved in ethanol to produce a 100uM stock solution stored at 4 oC.
Nile Red has a molar mass of 318.369 g/mol which is used to calculate the mass of
Nile Red needed to make a stock solution. Ethanol needs to be water-free as Nile
Red is not soluble in water. A magnetic stirrer is generally not needed as the Nile
Red dissolve quite readily into Ethanol. A scoopula can be used to mix the solution if
Protein samples are stained after aggregation is induced. The amount of Nile Red
added to protein solution is such so that the final Nile Red concentration is 5uM.

Preparation of Phosphate Citrate Buffer













Volumetric Flask
Magnetic Stirrer
Graduated cylinder
Glass Pipette

Stock solutions of 25mM citric acid anhydrous and 50mM sodium phosphate dibasic
heptahydrate are prepared. The stock solutions are mixed in the desired proportions
according Table 1. The final concentration of Phosphate citrate buffer is 37.5mM at
pH 7.
0.1Mcitric 0.2Msodium
0.1Mcitric 0.2Msodium
Concentratio acid(mL)
phosphate(mL) acid(mL),
If NaCl is added to change the ionic strength, the pH must be adjusted after the
addition of salt using a glass pipette. Ensure that the mixture is not stagnant by
using a magnetic stirrer.

Induction of Aggregation through Thermal Stress


IgG Solution


Laboratory Water Bath

15 ml Centrifuge Tubes
Tin Foil

Protein solutions are made at the desired pH and ionic strength. 3.5 ml aliquots are
injected into the centrifuge tubes and then placed in the Laboratory Water Bath for
20, 40, and 60 minutes at 60 oC. The water bath must have been running for atleast
an hour to allow the water to reach the desired temperature.

As soon as the samples have been heated for the desired time, they are
immediately transferred to a water bath at room temperature. Tin Foil can be used
as a cover for the beaker through which the tubes are poked. This ensures that the
samples remain vertical.
Samples are then centrifuged for 5 minutes at 400g and the decant is scanned
using fluorescence spectroscopy.

Spectrofluorometric Analysis
Instrument Settings for Fluorescence Spectroscopy
Intrinsic Fluorescence:
Measurements are collected at an excitation of 280 nm and an emission range from
290 to 400 nm. The PMT voltage is set at 600V and Slit Widths at 5 nm. The
scanning rate is set at 600nm/min.
Nile Red Fluorescence:
Measurements are collected at an excitation of 550 nm and an emission range from
570 to 750 nm. The PMT voltage is set at 600V and Slit Widths at 5 nm. The
scanning rate is set at 600nm/min.
Nile Red Staining of Solutions
Samples are injected into the cuvette along with the correct amount of Nile Red
stock solution. The cuvette is covered with a lid which is held down with one finger.
The cuvette is then inverted 5 times to ensure adequate mixing of the Nile Red with
the protein solution.

Daban, J. (2001). Fluorescent labeling of proteins with Nile red and 2-methoxy-2,4-diphenyl3(2H)-furanone: Physicochemical basis and application to the rapid staining of sodium dodecyl
sulfate polyacrylamide gels and Western blots. ELECTROPHORESIS Electrophoresis, 22(5),
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Demeule, B., Palais, C., Machaidze, G., Gurny, R., & Arvinte, T. (2009). New methods
allowing the detection of protein aggregates. MAbs, 1(2), 142-150.

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Protocols. Cambridge, MA: Humana Press.
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