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

Arthur Rowe arthur.rowe at nottingham.ac.uk
Fri Oct 22 06:35:01 PDT 2004


Apologies for the apparent assumption in my recent mail that there is only
one Arthur in the world!


Hi Yi-Ming and everyone

We do very large numbers of such analyses - i.e. defining how much aggregate
is present in a product. Generally we use the least-squares g(s) profile,
fitting multiple guassians (1 for each species), and following a standard
operating procedure to eliminate (as far as possible) subjectivity in choice
of resolution and other parameters. We use this approach rather than c(s),
as whilst with 'big monomers' the results will be identical, with lower s
species (I note that your monomer is only 6.4S) one can 'lose' some of the
n-mers in c(s) profiles.  It is also easy, fitting g(s) profiles, to
constrain your fit within sensible bounds for each n-mer species. Which is a
fair thing to do, if you know that only one  monomer species is present.

That being said, however, Hans-Joachim has a good point to make. Your 2
cells show the estimate for the amount of monomer to be 98 ± 1%. I doubt
that you can better that by much, however you do it! And your estimates for
the s values of  dimer, trimer & tetramer seem pretty good to me, given that
only tiny amounts are present. OK, on a good day, we expect to find the g(s)
profiles from 2 identically filled cells to be a little closer to each other
than your c(s) data might suggest. At the end of the day, it comes down to
whether you are doing an absolute estimate of amounts of n-mer, where
quality is all and time of little importance. Or whether what counts is
getting an answer to the question "how much aggregated?" to within a single
percentage point. Which you have achieved.

It's all a very big issue for the bio-pharma industry, of course.

Kind regards

Arthur


--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD   UK

Tel:        +44 (0)115 951 6156
           +44 (0)116 271 4502
Fax:        +44 (0)115 951 6157
email:      arthur.rowe at nottingham.ac.uk
Web:        www.nottingham.ac.uk/ncmh/business
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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



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