Pre-Treatment of analytical TSK-G300SWxl SE-HPLC column?

[Rande]

Friends,

I have been using TSK-G300SWxl (Tosoh Bioscience) columns for SE-HPLC of proteins, especially MAb's, for many years now. One recurrent problem has been the non-specific binding of some proteins - especially HMW peaks of aggregated IgG. When I have to "break in" a new column, it takes a long time and many injections until I can get reproducible results (% aggregate) with my reference standard.

(as shown in the D. Ejima, et al paper, the problem is not limited to this silica based column.)

Below are 3 different references that describe the problem and deal with it in different ways:

Who else has had this problem and how do you deal with it?

Thanx for your help ... all comments, suggestions or "food for thought" is welcome!


http://www.tosohbiosep.com/NR/rdonlyres ... SWXL01.pdf

http://www.tosohbiosep.com/NR/rdonlyres ... erSW01.pdf

Arginine as an effective additive in gel permeation chromatography
Journal of Chromatography A, Volume 1094, Issues 1-2, 11 November 2005, Pages 49-55
Daisuke Ejima, Ryosuke Yumioka, Tsutomu Arakawa and Kouhei Tsumoto

[HW Mueller]

Not yet having had the time to look at your refs. it can, nevertheless, be pointed out that TSK Gel columns have been discussed here for some years now, especially an unsolved problem that appeared with a TSKgelSuper SW 3000 column in this lab. In short, perfluoro acids permanently damaged its resolution power (before TFA, etc. I could almost separate a Mab from its Fab to baseline, afterwards there was strong overlap due to peak broadening, tailing).

Generally, one tries to keep the proteins very well solvated to prevent these absorptive effects. In my case I seem to remember the best results were obtained if they were dissolved in PBS (pH + ~7.2, ~0.15M in phos. buffer, 0.15M in NaCl) and the mobile phase was PBS. PBS didn´t do the trick after TFA, etc., though. The situation improved somewhat over time, but was not reversed entirely, even when trying different chaotropic mobile phases. I don´t remember trying a "hammer" like 6M urea. Also I never tried to inactivate possible active sites via any equilibrium of any substance that may do this, as I considered that too fragile.

One can not stress one point too often: The solvent of the protein sample which is co-injected plays a cruical role!