SEC impurity assay with a "sticky" enzyme

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

3 posts Page 1 of 1
Hi, I inherited an SEC HPLC method when I began working here a few years back. Recently, I have been trying to get ready to validate this method. Since we have no available impurities to spike with, I was going to use BSA as an impurity to spike our enzyme with. I immediately noticed that BSA and our enzyme co-elute in this system. However, our enzyme is ~150 kDa and I believe BSA is generally about 66.5 kDa which tells me that something is wrong. Our enzyme is known to be pretty "sticky" so I'm thinking that it is interacting with the SEC column in more than just a physical size exclusion sort of manner. Unfortunately, my previous HPLC experience has been mostly in reversed phase and small molecules, so I'm at a disadvantage trying to troubleshoot this method.

The mobile phase specified in the method is 100mM KH2PO4, 500mM NaCl, and 0.1% Tween-80 at a pH of 6.0. The column is a Tosoh TSKgel G3000SW XL 7.8x300mm, 5µm. I keep the column at 25°C and the flow rate is 1.0 mL/min. I detect using UV-Vis at 280nm if that matters at all.

I have done some experimenting with the mobile phase. Using 0.1% Tween-80 in PBS (which is about pH 7.6), BSA and our enzyme separate pretty well, however BSA elutes before our enzyme which seems backwards. As I understand SEC, the larger molecule should elute first, right?

Does anyone have any suggestions for me to try? Is there a different column or mobile phase I should try? I almost feel like I need to start over with this method, but I don't know where to start.

Thanks in advance!
does your protein have alpha and beta sub-units?
it could be breaking down as well on column and that is why it is co-eluting with BSA
can you try one of those proteins mixtures to see that all is fine with your column?

now remember one very important rule of the SEC you are doing:
you are not really separating by size. you are assuming that the Rh of your protein, being overall a sphere, goes by the rule that the bigger it gets, then the bigger the Rh gets and in almost the same ratio.
the SEC you are doing is separating accroding to Rh size.

it is possible to create denaturation of the protein, and this can impact the Rh and creates a "false size" for special cases.
Thank you for the reply. The protein does have heavy and light chains though it tends to be a very stable protein, especially in a buffer like our mobile phase. The information about the Rh is helpful. Like I said before, I am not very experienced with SEC so I was unaware of things like Rh. While our protein tends to be very stable, I'm sure that the pressures of HPLC could mess with it's Rh.

I am running a protein standard mixture right now to check that the column is ok. If that all checks out, could someone suggest a next step? How would one validate an SEC method with a molecule that possibly denatures on the column? Are there method parameters that can be changed to alleviate possible denaturation? Lower flow rate? Different mobile phase?
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