Chromatography Forum

Hello and good afternoon all!

Here is my problem. I am running a CEX method on a mAb using a Dionex WCX-10 column. When I initially ran the sample, my recovery was close to what i was theoretically expecting based on A280 UV data and the reported extinction coefficient of the mAb. However, as I ran more samples on the column, I noticed that my recoveries had dropped significantly and that I had begun to notice the emergence of a large injection peak which constituted an integrated area = to what I was missing. This seemed like the material was just not being retained so Im current trying to adjust the pH of my buffers. Any other suggestions?

Thanks in Advance

-Mant0

Most likely explanation is that your protein is denaturing (or otherwise degrading) in the sample solution.

To confirm or deny, make repeated injections of a single standard over a comparable time perios.

Tom,

Ive actually witnessed my loss in recovery over the course of several sequences run in the course of a week. In each sequence I did a minimum of 3 injections of sample.

In each sequence I did a minimum of 3 injections of sample.

and did the recovery drop with each injection? If it did, then it's a sample stability issue and adjusting your mobile phase won't help.

What else in in your sample? PBS etc...

Could it be that the column was not restored to original conditions after the initial injections?

In response to everyone, my sample is diluted in 1X PBS and I haven't seen decreasing areas with injections within a sequence. The losses I have seen have been on different days and each injection that day has been lower but consistent. As for the re-equilibration, I have a 15 minute re-equil time @ 1 ml/min. The column volume is approx 3 ml so this is roughly 5 column volumes.

Thanks again for your replies and sorry for the late one on my part. It was the weekend =p

Hi Mant0,

What would happen, if you extended the last part of the gradient? For instance, you could let 100 % B (the strong eluent) be effective for an hour or so – just to see whether or not you’re dealing with strongly retained analyte, or matrix, or whatever.

Best Regards

Danko,

I will give it a try and let you know how it goes.

Are you injecting the same sample when you compare the different days?

To Danko, I tried the long hold at 100% B to wash off anything still bound but it did not have any effect on my area recoveries.

To Uwe, I have done injections with the same sample (as in prepped and used on multiple days) as well as using a fresh sample for a new run and both showed the same recovery.

I have also tried adjusting the pH whcih did nothing as well. I'm wondering if the column has gone bad or whatnot. Although it is a new column.

OK, it would’ve been an easy fix, but it wasn’t that easy this time.

I’m almost convinced that you’re dealing with capacity decrease due to strongly retained stuff. The area increase of the injection peak is a clear evidence of that.
May I suggest the following experiment/test:?
Reduce the injected sample amount by half and then by 3 quarters etc. until your injection peak gets back to a normal size. It’ll hopefully confirm the idea of losing binding capacity and will give you a rough estimate of the scope of the problem.
The reason for not achieving any capacity restoration by the hold at 100 % B might be the elution strength of the B eluent. Maybe you’ll need to strengthen it (i.e. higher salt concentration and potentially addition of some organic solvent - IPA for instance)
Btw, what do your eluents consist of? What’s the pH?

Best Regards

There is something very "fishy" here, as it is claimed that injecting 3 samples, one after the other is ok, but next day there is this loss. Some mistake in shutting down, restarting?

danko,

Buffer is 10 mM MES (A) and 10 mM MES, 1 M NaCl (B) pH 5.75

Hi Mant0,

In that case, you should try adding some 15 – 20 % isopropanol to both eluents (A and B).
Did you try loading smaller sample amounts - as suggested earlier?

Best Regards

P.S. I also think that the story is a bit mysterious – as HW Mueller pointed out earlier. It may be a good idea to track down and confirm all the observations in this regard.