34 ChromatograPhy/SPeCtroSCoPy
➠
SLS measurements of molecular weight, known to All three samples show a peak at around 10 nm
be in the region of 145 kDa, support this view. The (Fig. 3), the expected diameter of the antibody, but the
measured molecular weight for duplicate untreated relative width of this peak and the size distribution is
samples is slightly low (129 ± 21 and 139 ± 8 kDa), a different for each sample. With those samples stored
result attributable to the presence of antibody fragments under non-ideal conditions, there is evidence of large
not removed in the purification process. The results from aggregates, particularly where subjected to freeze/thaw
duplicate treated samples on the other hand have a much cycles. The ‘quality’ of each sample is evident from
higher molecular weight of 1210±125 and 1270±65 kDa, z-average diameter and polydispersity index data, which
providing further evidence of aggregation. show that freeze/thaw cycling is particularly detrimental.
Displaying size data in terms of a volume distribution
highlights the sensitivity of DLS. Even for the sample
subjected to the worst conditions, the volume distribution
shows no evidence of aggregation (Fig. 4) because the
percentage volume contribution of the main protein peak
is still 99.6 per cent. This means that the distribution
by intensity is extremely sensitive to changes in the size
and aggregation state. Because of this DLS is a powerful
technique for detecting the presence of very small
numbers of relatively large particles, which can give an
early indication of stability issues during protein storage
Fig. 3. Size distribution (by percent intensity) from antibody samples
or during a treatment process.
stored under different conditions: 4°C (blue), 25°C then 4°C (red) and
Static light scattering results for the sample stored
5x frozen/thawed (green).
exclusively at 4°C reflect the expected molecular weight
for IgG, with a measured value of 150±13 kDa. The
A temperature scanning routine embedded in sample stored under warmer conditions has a higher
the Zetasizer software permits the study of size and molecular weight, 176±6 kDa, indicating a degree of
scattering intensity as a function of temperature. A aggregation or oligomerisation. The third sample however
protein ‘melts’ under the influence of heat when the also has a measured molecular weight close to the
molecules denature – leading to massive aggregation. expected value, 156±12 kDa. In this case aggregates
This transition is visible in light scattering studies as a are present at a level at which the Zetasizer’s advanced
significant increase in both size and scattering intensity. dust rejection algorithm can be used to remove the
The untreated samples exhibit this behaviour at a contribution of the large particles. This algorithm allows
temperature of around 56°C (Fig. 2), the melting point the measurement of molecular weight of the protein,
for the antibody. With the treated sample no transition is despite the presence of some large aggregates.
observed; the treatment process has already permanently
denatured the antibody.
Table 1. Size and polydispersity index are influenced
by storage conditions.
Storage Condition Z average Pdi
(diameter, nm)
35 days at 4°C 11.4 0.075
31 days at 25°C,
11.7 0.172
then 4°C
5 freeze/thaw
152 0.646
cycles, then 4°C
In conclusion, then, light scattering techniques provide
Fig. 4. Size distribution (by percent volume) from antibody samples
rapid access to accurate molecular weight and size
stored under different conditions: 4°C (blue), 25°C then 4°C (red) and
information for the characterisation of proteins. With
5x frozen/thawed (green).
the Zetasizer, measurements of these and other sample
To investigate the effect of storage conditions on IgG, properties are straightforward, even with small sample
freshly prepared antibody samples were treated in the volumes and low concentrations. These are extremely
following ways: stored for 35 days at 4°C; stored for 31 important considerations for the large majority of
days at 25°C, then for four days at 4°C; and subjected to pharmaceutical and biomolecular applications. u
five freeze/thaw cycles then stored at 4°C.
All samples were prepared at a concentration of Ulf Nobbmann is Specialist, Nanometrics, with Malvern
0.8 mg/ml in an ammonium hydrocarbonate buffer, at a Instruments, Malvern, England, For more information, visit
pH of 6.8. www.malvern.com
www.scientistlive.com
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