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European Biopharmaceutical Review
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Bob Carr and Andrew Malloy at NanoSight Limited discuss the direct visualisation, sizingand counting of aggregation in proteins through the use of nanoparticle tracking analysis
Characterising the state of aggregation in proteins is of paramount
importance when trying to understand biopharmaceutical product
stability and efficacy. Product quality, both in terms of biological
activity and immunogenicity, can be highly influenced by the state of
protein aggregation.
A wide variety of aggregates are encountered in biopharmaceutical
samples, with a range of sizes and characteristics (for example,
soluble or insoluble, covalent or noncovalent, reversible or
irreversible). Protein aggregates span a broad size range, from small
oligomers (nanometres) to insoluble micron-sized aggregates that can
contain millions of monomer units. Protein aggregation can occur at all
stages in the manufacturing process (cell culture, purification and
formulation), storage, distribution or handling of products. It results
from various kinds of stress, such as agitation and exposure to
extremes of pH, temperature, ionic strength, or various interfaces (for
example, air-liquid interface). High protein concentrations (as in the
case of some monoclonal antibody formulations) can further increase the
likelihood of aggregation.
Aggregation therefore needs to be carefully characterised and
controlled during development, manufacture and subsequent storage of a
drug substance and formulated product. Similarly, by monitoring the
state of aggregation, modification and optimisation of the production
process can be achieved.
The technology to handle this application is provided in a new
laser-based system applying nanoparticle tracking analysis (NTA). This
allows nanoscale particles, such as protein aggregates, to be directly
and individually visualised and counted in liquid in real-time, from
which highresolution particle size distribution profiles can be
obtained. The technique is fast, robust, accurate and low-cost,
offering an attractive alternative or complement to existing methods of
nanoparticle analysis such as dynamic light scattering (DLS) (also
known as photon correlation spectroscopy (PCS)) or electron microscopy.
IMAGING PROTEIN AGGREGATES
NTA offers a unique insight into protein aggregation in the range
of 30nm to 1,000nm. Having visually inspected the sample for the
presence of aggregated material (see Figure 1), the user can rapidly
generate a particle size distribution profile and count (in terms of
aggregate number concentration) of the aggregates seen (see Figure 2).
COVERING THE SIZE RANGE
Historically, a number of techniques have been used to characterise
proteins and protein aggregation. Separation techniques are often used
to discriminate proteins and protein aggregates, with further analysis
performed on the separated sample.
Analytical techniques include:
- Dynamic light scattering (DLS)
- Multi-angle light laser scattering (MALLS)
- UV spectroscopy
- Light obscuration
- Micro-flow imaging (MFI)
- Nanoparticle tracking analysis (NTA)
Separation techniques include:
- Size exclusion chromatography (SEC)
- Field flow fractionation (FFF)
- Capillary electrophoresis (CE)
- Analytical ultracentrifugation (AUC)
Sub 30nm
It is common to find SEC paired with DLS, MALLS or UV spectroscopy.
Size exclusion chromatography can be used to separate protein monomers
from aggregates. Subsequent analysis using DLS, for example, can
produce accurate size and molecular weight analysis for purified
fractions. There is no separation above the exclusion limit of the SEC
column, and as such bulk analysis systems such as DLS become less
well-suited. MALLS analysis can help reduce the effect of larger
aggregates in nonfractionated samples, but the technique requires
interpretation.
30 to 1,000nm Range
NTA allows protein aggregates within the size range of 30 to 1,000nm to
be individually imaged and sized by tracking their Brownian motion on a
particle-byparticle basis. This form of analysis allows high resolution
number distributions to be generated. This region is often poorly
served by DLS, with a high concentration of protein monomer and a low
number of large, bright aggregates often dominating the signal. While
fractionation can be performed, for example with FFF to aide DLS
analysis, the dilution that is often required for FFF can make this
route undesirable due to the potential for further aggregation.
Furthermore, dilution of these ‘mid-sized’ aggregates often takes them
below the concentration sensitivity limit for DLS. The technique
frequently requires no dilution as the 30-1000 nm protein aggregates
often fall within the optimum concentration range for this experimental
protocol.
The cut-off limit of the technique (approximately 30nm for protein
aggregates) means that it is well suited to complement SEC/DLS or
SEC/UV above the exclusion limit of SEC. The upper limit of the
technique represents the point at which conventional single particle
imaging and obscuration techniques become applicable.
With no prior separation of aggregates, DLS would typically produce a
bimodal result for the aggregated sample shown in Figure 3.
The primary peak would be formed from the large number of monomeric
particles, while the secondary peak would be formed by very large
aggregates which scatter significant intensities of light. A poorly
resolved DLS analysis would show no particles between these points
despite their existence as the primary monomeric particles and the few
larger aggregates would dominate the signal.
While NTA would be unable to measure the primary monomeric size as the
particles would fall below the detection limit of the technique, above
30nm, the technique provides particle-by-particle analysis of protein
aggregates, uniquely forming a high-resolution number distribution of
aggregated particles.
Shear Stress
In this example, a virus was correctly measured by NTA at 45nm diameter
(see Figure 4a). However, following agitation of the same
sample by shaking it for a few seconds, shear stress was seen to have
induced aggregation in the virus sample. Note
the change in scale of the normalised vertical axis shows a drop in
particle concentration on aggregation (approximately 80x106 particles per millilitre to approximately 50x106 particles per millilitre). Such information is unavailable to other ensemble light scattering techniques such as DLS (2).
Heat Stress
In this example, a sample of IgG was heat stressed at 50ºC
for 35 minutes in the sample chamber and the aggregation followed in
real-time using the batch capture facility in the NTA programme. In
Figure 5, the size distribution (middle panels) with the
corresponding NTA video frame (left panels) and 3D graph (size versus
intensity versus concentration; right panels) are shown.
Aggregation Following Dilution in Different Quality Waters
In this example, two nanoparticle sample types (chitosan nanoparticles
and gold calibration nanoparticles) were diluted in waters of varying
quality.
- Tap-water (from a hard-water area)
- Deionised water (for use in batteries)
- High purity, reagent grade (18MΩ) water
A) Chitosan Nanoparticles
Firstly, samples of chitosan nanoparticles (a bioadhesive
polysaccharide) developed for use in drug delivery applications
(supplied by IPATIMUP – Instituto de Patologia e Imunologia Molecular
da Universidade do Porto) were diluted in the three water types shown
above and the size of population measured immediately on dilution and
after five to 10 minutes. The effect of reduction of ion and mineral
content in water on aggregation can be clearly seen in Figure 6.
B) Gold Nanoparticles (NIST Standards)
Calibrated 30nm gold particles (NIST) were diluted in the same three
types of water: tap, de-ionised and 18MΩ water (all free from
nanoparticles) then analysed with the same concentration using NTA.
Figure 7 shows that the degree of aggregation depends on water purity,
with only the pure 18MΩ water causing no aggregation.
References
- Filipe V, Hawe A and Jiskoot W, Critical Evaluation of
Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement
of Nanoparticles and Protein Aggregates, Pharmaceutical Research, 27(5): pp796-810, 2010
- Moser M, Emerging analytical techniques to characterise vaccines, Proc Intl Conf Vaccines Europe, December 2008
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