Chromatography Forum

Nice that we finally get a piece of vital information, a protein with a detergent stabilizer is not the same as a protein without that. There will be a tremendous incompatibility arising as the protein-detergent hits the mobile phase. I would think that the mobile phase either has to contain the right concentration of that detergent or, if the column is incompatible with this detergent, that it is removed prior to the chromatography. In the latter case one has to find some protein solubilization substance which is compatible with the chromatography and the protein.
I think it was in the example I mentioned above of separating Mab from its Fab that required exchange of sample buffers to the one best for the SEC, otherwise there was a horrendous distortion of peaks. (The samples were ultrafiltered, the retentate washed with SEC mobile phase buffer via filtration, then the protein retentate removed from the filter with the SEC buffer).

Hi

I also run a membrane protein solubilised in detergent (DDM - the same as DaveM) on a Tosoh SuperSW3000. I use this set-up for rapid screening of different proteins in different detergents. In each run I have a protein of interest (POI) with a fluorescent tag attached. This allows me to detect two chromatographic readouts, one for protein (uv280nm) and one for fluorescence. I do this because my POI is solubilised from whole cells incubated in detergent, then ultra-centrifuged to spin out debris and the cell solution is run on the column. From this I get a dirty protein signal from the protein mix in the cell solution and a specific fluoresence signal that corresponds to my POI. This means I can see my protein as pure fluorescent signal in the noise of a messy protein signal (this is great for rapid screening as it avoids large-scale purifications).

PROBLEM: I notice that the first time I run cell-samples on a new column they give fantastic single-peak fluorescent signals for my POI but the peak signal deteriorates rapidly between each set of runs, say one run of 15 samples each week. After just a few weeks (and 50 sample injections) my fluorescent signal has deteriorated by over 90 %. It is not a separation/resolution issue as the small residual peak is still single and sharp. No amount of cleaning with ethanol or methonal or various salts seems to help.

What is strange is that if I produce my POI on a large-scale and purify it so it is completely pure, then run it on the same deteriorated-column in detergent but without the cell-mix then the protein signal is fine. (Although after a last-ditch wash of the column in 0.1% SDS this signal stopped working as well).

Obviously the whole point of the screening is be able to cheaply and rapidly test many proteins which is not possible if I have to keep replacing the column. If anyone can explain what is going on or suggest how to avoid the deterioration in screening I would be immensely grateful.

Cheers, Paul

ps - I used a new guard-column for this but still got the deterioration.

Danko:
From what I've seen since I made that post I believe the column was on it's last legs. We replaced it today and there was a dramatic improvement. Based on the symptoms, my hunch is that there was a dead space at the top of the column. My retention times were increasing gradually, split peaks or shoulders, increasingly broad peaks, and whereas previously the uv signal dropped to zero within 15 minutes, it'd started taking almost thirty to settle down.

Peak splitting can also be caused by a dirty column inlet. A colleague of mine has always preferred a particular column manufacturer for working with proteins. He have said something, that the inlet frits of this manufacturer are different from other ones and works better with proteins. Unfortunately I can not even judge, because I've always worked with other analytes.

Do you remember the manufacturer that he preferred?

Welcome back, Gerhard!

Some of you who have been around for some time might remember that I had a permanent deterioration of resolution in a Tosoh Super SW after it was exposed to TFA. I never found out what caused it. These columns seem to be very touchy, but their performance is really super. I almost completely separated a monoclonal antibody from its Fab fragment (after the TFA there was a bit more overlap), while a German manufacturer of polymer SEC columns got a, as they called it, "separation" of the Mab and its Fab in the form of widening of the peak (the mixture of Mab and Fab gave a peak slightly broader than the Mab alone, no sign of two peaks, whatever).

Hi Mueller, can I ask what is the mobile phase you used for the Fab fragment analysis. Maybe you will be interested in this data: http://www.sepax-tech.com/application_notes/ZM1005.pdf

Various mobile phases were tried, with different results, its a complicated story. The best was PBS. The solution used to dissolve the Mab and Fab also had a strong effect on the chromatography.

Dear DM.
I worked in labs that performer 24/7 LC of the same methods on the same column types for years. We knew our methods and columns e.g. UPLC last up to 5 K injections. But it is not exactly 5,000. It does not mean that 4,999th injection is OK and the next one will fail SS. As batches are slightly different, one may have this month more samples of liver, next period more samples of muscle. But we knew, that around 5K is what to expect. If you just started you will not know what to expect, consult with performance of similar methods of others. The key factors here - good clean up, good wash and good column care.
Alexandre

Do you remember the manufacturer that he preferred?

If I remember correctly, he used the columns from "Dr. Maisch GmbH". But remember please, this comment is not based on my personally experience. I have never worked with proteins.