[RASMB] precision limit in quantifying protein aggregates by sedimentation velocity with Sedfit

[Yi-Ming_Li at hgsi.com]

Hi! Everyone:
We routinely use Sedfit to analyze sedimentation velocity data to detect
the aggregates in our protein products. Many of our results are consistent
with the size-exclusion HPLC regarding the percentages of the aggregates in
protein products. But we also found that sometimes the repeatability of
sedimentation velocity analysis is not very good. For example, in order to
test the repeatability of the method, we loaded the same sample (a
monoclonal antibody) to two different cells and make a sedimentation
velocity run. Then we analyzed the data from the two different cells using
sedfit to get C(S) profiles. From the C(S) profiles we calculated the
percentage of the species. We got the following results:

Cell 1:
99.07% @ 6.4 s
0.74% @ 9.7 s
0.1% @ 11.3 s
0.08% @ 16.9 s
rmsd: 0.003517

Cell 2:
97% @ 6.4 s
1.43% @ 8.9 s
0.42% @ 10.9 s
0.27% @ 13.6
0.15% @ 18.2
rmsd: 0.003533

According to Dr. John Philo, sedimentation velocity is an excellent method
to detect and quantify the aggregate in protein products. Therefore I think
that the variations here are beyond the range we expect. Moreover, the
sedimentation coefficient of the dimmer and other oligomers are quite
different. Could anyone tell me, based on your experience and knowledge,
the precision limit of the software for quantitative determination of the
percentage of protein aggregate in a non-interactive system? If the
precision limit of the software is not a problem, what are the possible
sources for such large variations? Is it possible that the variations came
from the instrument or centerpieces?

Thanks!

Yi-Ming