Chromatography Forum

Hi everyone,

I am currently using a mixed mode resin (capto adhere from GE) trying to separate an antibody like protein from aggregate. I get very severe Peak tailing on this resin unsing it in bind and elute mode. (HETP and As look normal with acetone). I was wondering what causes this. It almost looks like the peak is becoming asymptotic to the x-axis. Could the problem be a Freundlich isotherm like behaviour of the protein?
Has anybody had any similar experiences?

Thanks for the help!
S

My understanding is that the Freundlich isotherm curves up at higher concentration. This would produce fronting, not tailing.

I would guess that your elution buffer is a little weak and your peak is “slowly bleeding offâ€

Hi, thanks for the answer,

did you mean "Capto MMC" (with a C) ? or is there another MM resin from GE that I am not aware of? The other resins you mention are Hydrophobic charge induction resins, I think. As I am ultimately aiming at using the resin in a displacement mode, I am not sure if those are suitable. But I will have a look. What do you think?

Thanks again,
S

did you mean "Capto MMC" (with a C)

Stefan,

Yes, it looks like that was a typo - I was refering to Capto MMC. I have not worked with this resin, but from the literature available - it seems similar to the MEP HyperCel (Hydrophobic charge induction resin).

I am ultimately aiming at using the resin in a displacement mode

Do you really mean "Displacement Chromatography"? Such as eluting with a charged polymer like dextran sulfate or a highly charged ion (or do you mean "flow through mode")? Displacement Chromatography is very interesting, and I have played with it a little. Unfortunately, most of the displacement chromatography I have done, was by accident with very unexpected results.

Quick story from a previous job: Purification process for a monoclonal antibody with minimal development before scale up. Initial capture was on protein A with citrate elution buffer. Next step was polishing on Q sepharose. We decided that instead of a buffer exchange, we could just dilute the protein A elution pool and it would bind to the Q. This worked OK at small scale with a light load, but on scale up - we had a sudden elution peak during the product load. This was lost into the large volume of flow through waste. We later determined that during loading, both citrate and antibody were binding to the Q resin. When the resin got close to capacity – the citrate had a stronger affinity and displaced the protein. There is a reason why you are supposed to use a cationic buffer with anion exchange resins!!

Sorry if I got side-tracked from your original question, but hopefully someone will learn from my mistakes. That is what a forum like this is all about.

Rande, what´s a cationic buffer?

Hey Rande,

thanks for your answer...very interesting. Yes I am in fact trying to use different resins in displacement mode. Real Displacement chromatography. We are currently getting some displacers from Sachem.

But I cannot yet solve the problems with Capto adhere. Additionally, my protein of interest seems to be precipitating onto the resin in batch mode...as long as the guys from GE I asked for assistance don't come up with any useful tips any time soon, I fear I hit a dead end

what´s a cationic buffer?

Hans,
What I mean is : a buffer system where the buffering ion is positively charged (such as Tris) as compared to an Anionic buffer such as carbonate, phosphate or acetate. I have heard this terminology used often when talking about ion exchange chromatography, I am not sure it is accepted scientific nomenclature.


Stephan,

I was looking at the Sachem Web site. It looks like interesting stuff, but I am not sure what the advantages are for your application. If you are aiming to purify a pharmaceutical product under GMP, I am always afraid of being the first to present new technology to the FDA. Might be interesting science – might be a better process – but you will get too many questions from a reviewer.


If you are looking for a polishing step to remove a low level contaminant, have you looked at the charged membrane filters (such as from Millipore, Pall or Sartorius) ? These work well for flow through – final polishing. They have many advantages for manufacturing, such as “no column packingâ€