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

[Steve Shire]

Yi-Ming,
      Yes we have made similar observations. We are in the process to  
determine if this is a problem specific to our instrument or whether  
this is a limitation with the current status of the technology. I am  
away from Genentech until Oct. 27, and will try to get back to you with  
additional details after my return.
             Steve
On Oct 21, 2004, at 4:07 PM, Yi-Ming_Li at hgsi.com wrote:

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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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