Chromatography Forum

danko,

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

Your cleaning eluent is not nearly aggresive enough. If your contaminants are basic proteins with pIs above 10 or even 9-and many MAb isoforms fall into that category-then at pH 5.75 they are very tightly stuck to the column and 1 M salt may not budge them. Dionex recommends up to 2 M salt and, for severe contamination, up to 500 micro L injection of 1 N NaOH. Initially I would initially recommend at least 1 M salt pH 11 for 10 col vols. I think you may be pleasantly surprised at the improvement.

Hmmh... I am not sure that a column contamination can explain a partial (!) breakthrough of an analyte. Also, if I want to clean a WCX column, I would go to strongly acidic conditions to eliminate the charge on the ion exchanger. At alkaline conditions, you are still left with analytes with a permanent positive charge that still stick to the packing. To me, partial breakthrough does not sound like a problem with the column, but like something that has to do with the sample - age, preparation etc. This is why I asked the questions about the sample, but I am still missing something since the problem seems to remain unresolved.

Hmmh... I am not sure that a column contamination can explain a partial (!) breakthrough of an analyte. Also, if I want to clean a WCX column, I would go to strongly acidic conditions to eliminate the charge on the ion exchanger. At alkaline conditions, you are still left with analytes with a permanent positive charge that still stick to the packing. To me, partial breakthrough does not sound like a problem with the column, but like something that has to do with the sample - age, preparation etc. This is why I asked the questions about the sample, but I am still missing something since the problem seems to remain unresolved.

That may be good advice if one is dealing mainly with small, permanently charged molecules, but it is well off the mark with a complex mix of proteins. The surface of a typical monoclonal antibody is a complex patchwork of hydrophobic surface, negatively charged (mostly carboxyl) groups and positively charged nitrogen groups. These groups are all titratable. While it is true that at very acidic pH you will greatly reduce the charge of the column, you will also make it and the protein more hydrophobic and reduce greatly reduce the powerful electrostatic forces that could push the protein off the column by making the protein ever more positive, and thus still attracted to any residual negative charge on the column. In contrast, at high pH the net charge on most contaminting proteins would become highly negative while the column is highly negative also and the resulting repulsive electrostatic free energy term would become very large. That is why Dionex recommends removing really stubborn protein contaminants with NaOH. That said, following such a protocol with a low pH elution in the presence of a compatible organic such as ACN is certainly a good idea

When I was suggesting an acidic wash, I was not thinking about proteins as the only possible column contaminant, I was thinking about other gunk from the matrix that might stick to the column. But this is neither here nor there. I continue to be puzzled by the idea that you can get retention AND breakthrough of the SAME species. The only time I have seen something like this is when the elution speed was too high to get complete interaction with the matrix. I would be surprised to find such effects on a packing designed for this type of separations. In the absense of this effect, the only other explanation that I see is that the analyte consists of different entities that change during aging or due to other causes. Of course, large proteins are complex beasts and often by no means uniform.

We are obviously not told some essential(s), or I don´t understand something. What I understand: The column results are as expected at the beginning of different days, but consecutive injections during the same day show an ever decreasing peak with the difference showing up as breakthrough.

How can a Mab deteriorate within minutes it takes to do consecutive runs, but then recover over night???

Then if it is the column, how does the column recover over night??

Regarding ionic interactions with silanols I have seen many times breakthrough and retention occuring simultaneously, but as more or less a single very broad peak. Thus, I also have trouble understanding how one can have one part come out at tm (to) the rest normal, just smaller, with nothing inbetween.

One thing that comes to mind is a variable concentration of salt in the sample, potentially together with an additive that creates a high viscosity. Double peaks of the type described here are observed in reversed-phase chromatography of small molecules when the analyte is injected in DMSO, which is both a strong solvent and has a strong viscosity maximum with water. You get a plug of analyte in a strong eluent that runs through the column unretained and does not release the analyte, or only a portion of it.