chromAtogrAphy/spectroscopy 33
Assessing the quality of proteins
using light scattering techniques
P
roteins consist of polypeptide chains
dynamic, static and electrophoretic light
Ulf Nobbmann uses two
that are sensitive to a wide range of
scattering measurement capabilities.
case studies to illustrate
parameters such as temperature and
Fig. 1. shows DLS data for treated and
the practical application
chemical environment. Preparation
untreated samples of a therapeutic antibody
of different light
method, storage conditions and/or buffer choice
supplied in an ammonium phosphate buffer.
scattering techniques. can all influence the size and quality of proteins
Details of the associated treatment process are
in a sample.
unknown.
Understanding the impact of different
Ulf Nobbmann utilise
The samples are clearly different. The treated
variables on a given protein is essential both for
deux études de cas pour
sample contains particles with a diameter of more
the production and maintenance of high quality
illustrer l’application
than 50 nm whereas the z-average size of the
pratique de différentes
materials.
untreated sample is around 11 nm, the expected
techniques de diffusion de
Light scattering techniques are proving ideal
value for the antibody.
la lumière.
for protein analysis as
they deliver data on both
size and molecular weight.
Ulf Nobbman illustriert
Dynamic light scattering
anhand von zwei
(DLS) exploits the link
Fallstudien die
between hydrodynamic
praktische Anwendung
von unterschiedlichen
particle size and
Lichtstreuungstechniken.
diffusion rate, calculated
from measurements of
time-dependent light
fluctuations.
Static light scattering
(SLS) involves the Fig. 1. Size distribution from pure (red) and treated (green) antibody
determination of molecular sample.
weight from measurements
of
time-averaged intensity
at a range of sample
concentrations. Together
the techniques provide
complementary insights
into the factors influencing
protein quality.
Here, two case studies
illustrate the practical
application of light
scattering techniques.
The first compares
the properties of two
therapeutic antibody
samples, one a treated Fig. 2. Melting point determination of the untreated sample. TM = 56°C.
analogue of the other.
The second study is a controlled investigation Visually, the broad peak shape of the treated
of the impact of storage conditions on sample suggests the presence of oligomeric
immunoglobulin G (IgG). Both cases reference assemblies and/or aggregates. The associated
data generated using instruments from the polydispersity index is greater than 0.1, indicating
Zetasizer family of particle characterisation the presence of more than one species. These
systems (Malvern Instruments), the first results suggest that the treatment process has
commercially available instruments to combine promoted aggregation of the antibody.
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