[RASMB] Precision of c(s) method
Tom Laue
Tom.Laue at unh.edu
Fri Mar 24 05:08:51 PST 2006
Hi Matt-
This is an excellent, but difficult, question. I expect there will be a
good discussion. Here's my two cents:
Sedimentation velocity is a technique of *accuracy* and not merely
precision. The x-axis of a c(s) plot has real physical meaning, and the
data from an experiment stand on their own (no standards are needed).
Size exclusion is a technique of precision- reproducibility is all that
is required, and the reproducibility is relative to a set of standards.
Hence, SV and SEC are truly orthogonal methods.
Since SV is a technique of accuracy, reproducibility depends on
everything being the same. While much of an experiment is under your
control, and thus can be made reproducible, some aspects are hard for
you to control. For example, small differences in centerpieces and how
they are assemble can make a difference. Certainly the reproducible
alignment of the centerpieces is important. Most importantly, c(s) is
curve fitting and can ONLY tell you about precision and not accuracy.
Changes to the fitting parameters will affect the result returned by the
analysis.
That said, careful work typically can get you to the 1% range.
If you want to be sure that you have 1% of an aggregate (an accuracy
question), however, you must do repeated experiments- you will never be
able to be certain by reanalyzing the same set of data (which can only
tell you about precision).
Best wishes,
Tom
Parker, Matthew wrote:
> Hi folks.
>
> I was wondering if people could give their views on the degree of
> precision that can be expected from the c(s) method, or other methods
> for fitting SV data, for assessing the degree of aggregate formation
> in a protein sample. (Monoclonal antibodies, in particular.) The FDA
> is starting to ask for “orthogonal methods” to back up SEC data on
> aggregate formation. Is it reasonable to expect that SV can give the
> same degree of reproducibility when dealing with, say, 1 to 5%
> aggregated species? Has anybody done any systematic determinations of
> the precision of the method? (Subjective impressions are also welcome!)
>
> Thanks,
>
> Matt
>
> Matthew Parker, Ph.D.
>
> Analytical and Pharmaceutical Sciences
>
> Immunogen, Inc.
>
> 128 Sydney St.
>
> Cambridge, MA 02139
>
> (617) 444-2028
>
> (617) 995-2544 fax
>
> matthew.parker at immunogen.com <mailto:matthew.parker at immunogen.com>
>
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--
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX: 603-862-0031
E-mail: Tom.Laue at unh.edu
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