rapid deterioration of SEC column performance

[oldtimer]

We had been using TSK and Water's Biosuite 250 columns successfully for the past 2 years for many antibody compounds with great success, but recent lots of columns are failing ( excess monomer tailing, loss of resolution, etc) after just a few runs. Mobile phases are typical phosphate buffered saline of various strengths and pH ( 0.1M phos, 0.15M NaCl at pH 6.8 typical mix). Test mixtures like used by manufacturers perform well on these bad columns, but the antibodies profile badly. We've even returned some columns to the manufacturer for troubleshooting, but all their routine tests suggest columns are OK. This column failure is affecting multiple projects at multiple sites (US and OUS) to where people are questioning the robustness of SEC, in general.

Any thoughts on these sudden performance failures for antibodies and/or on better test mixtures to indicate acceptabe columns for antibody SEC?

[Kostas Petritis]

Are you saying that columns that used to work for a particular antibody analysis are now "failing"? If this is the case, I would doubt that something changed in the production of these columns from the manufacturer. Probably you are doing (or not doing any more -such as passivation?-) something different that result in this column "failing".

[HW Mueller]

Definatly one should first look at changes that have creeped in regarding mobile phase, sample solvent, inappropriate use of columns by others, rather than the column itself.
To me it sounds like a change in the matrix (solvent, etc.) of your antibody samles.

[Mattias]

We had a similar problem with the Superdex SEC columns. Typically, the SEC columns have many different modes of action (SEC of course, but also some reversed-phase and ion-exchange). The supplier only tested the SEC properties in their test chromatograms, by using a lot of salt and organic in the mobile phase. The reversed-phase properties were not tested at all - and it can have a big importance for peptides and proteins.

We complained about strange retention times (some peptides started to elute several minutes after the exclusion volume!). The supplier only said that all columns were within spec...

[oldtimer]

Thanks for inputs so far. We have 10 different compounds using 10 validated methods with years of proven use and robustness testing. Columns are dedicated to each project and are carefully maintained by experienced chromatographers running these methods in multiple labs around the world. Suddenly, instead of the routine quick equilibration right out of the box that we're used to, we fail either right away or shortly after routine use.

Since these are rugged, proven methods with long histories of support by multiple users, I doubt that suddenly every reagent source has gone bad or the experienced chromatographers have had some sort of melt down at the same time. We obviously have 1 common denominator: columns.

[HW Mueller]

It is extremely unlikely that only you get bad columns. If the column manufacturers suddenly were unable to make these, others would have the same trouble also. I would look at differences regarding the samples themselves, maybe someone found a way to improve something in the antibody prep......

[Spruce]

You are not the only one! We have had problems with the TSKgelG3000SWXL for almost a year from now, bad resolution and some artefact peaks and I know at least one other company having problem with TSK. Some columns are working but not everyone. We are looking for some other columns because the supplier can´t admit any changes in manufacturing.

[HW Mueller]

Could be that the problems are due to something akin to what I have mentioned here for years, without ever finding an explanation. The resolution of a TSK went way down when using perfluorinated acids in the mobile phase. It recovered only partially after running for quite some time on PBS.