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

Schoenfeld, Hans-J. {PRBD~Basel} hans-j.schoenfeld at Roche.COM
Fri Oct 22 04:11:03 PDT 2004


Dear Yi-Ming,
You try to quantify details within less than 3% of aggregates that are present beneath more than 97% of monomers. Regarding the facts that accuracy of AUC may be estimated to be better than 5% and that the signal (UV absorption) obtained from the aggregates contributes to the overall signal in a linear way regarding mass, I assume that you cannot expect a higher accuracy from this method than you obtained.

A more accurate measurement of small quantities of aggregates beneath big amounts of non-aggregated protein would probably be obtained with light scattering methods (I suggest dynamic light scattering for your application). Here the signal, which is the intensity of scattered light, is proportional to the square of mass. Therefore, you would obtain far more signal from the aggregates than from the monomers. Problems with this method are right the opposite: quantification will be difficult when aggregates are too many or too big compared to the monomer fraction.

Size-exclusion HPLC may influence the actual size distribution of your samples when equilibria play a role or materials get absorbed (e.g. in the entrance filter of your column).
Good luck and best regards,
Hans-Joachim Schönfeld.

>============================================
>Dr. Hans-Joachim Schönfeld
>F. Hoffmann-La Roche Inc.
>PRBD-E, B93/5.44
>CH-4070 Basel
>Switzerland
>
>Tel. (+41) 61 688 28 95
>Fax. (+41) 61 688 90 60
mailto:hans-j.schoenfeld at roche.com


-----Original Message-----
From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org] On Behalf Of Yi-Ming_Li at hgsi.com
Sent: Friday, October 22, 2004 1:07 AM
To: rasmb at server1.bbri.org
Subject: [RASMB] precision limit in quantifying protein aggregates by sedimentation velocity with Sedfit


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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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