Hi Wei:
We have been using RP-HPLC/MS for mAB characterization for the last few years.
150 KDa protein is too large for RP-HPLC separation. Instead, we reduce the mAB
to light Chain (MW = 23 KDa )and heavy chain (50K) then use RP-HPLC/MS for mass
measurement. We have found that PLRP column with pore size 4000A and particle
size 8 u is the best for large proteins. By using this column, we see very
little carry-overs. You can order this column from Michrom Bioresource
(530-888-6498) in California.
What is the % of the minor peak in your protein? Is this peak a mAB too? If it
is not a mAB, it is much easy to deal with now. You can use Protein A draping
column to separate the mAB from the minor peak and enrich the minor peak by
vacuum dry the flow through from protein A. If the unknown peak is a product of
posttranslational modification of your mAB, you may still able to see this peak
in mass spectrometer even it is not seperated from the mAB. This will depend on
what is the modification and what kind of mass spectrometer you have.
Good luck with your characterization.
Rong-Rong Zhu
BASF Bioresearch Corp.
508-849-2770
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