We are replacing SDS-PAGE with CE-SDS in our lab. In developing a Non-reducing CE-SDS method for one of our monoclonal antibodies, we are getting a small peak at the expected MW of ~ 146kDa, however there are two larger bands at 200kDa and ~ 220kDa. We are looking into longer heating times and higher %SDS conditions in case this is due to incomplete denaturation. Are there any other reasons this could be occurring?
Hi! In principle glycosylations can cause such problems, as SDS cannot attach to the sugar moieties and hence analytes are slower ie appear larger. Which gel are you using? Would it be feasible to include two standards, ie eg ovalbumin and thyroglobulin to adjust electropherograms? Have you tested also other antibodies and are these results closer to the expected ones?
We are using the Beckman SDS-MW gel. We have analyzed a couple of our other mAbs and they also have higher than expected MW when calibrated with the Beckman standards. We are looking into making our own standard based on Zhang et. al JPBA 2010 and hope that will work better. I will also suggest the second standard. Thank you.
Hi Shannon, what you could be seeing is the different glycosylation forms of your antibody.
If this is the case, then you should see a difference if you treat your Ab with PNGase F
https://www.neb.com/products/p6039-protein-deglycosylation-mix
thank you. We will be performing the PNGase F treatment. However we don't expect to see such a large variation in MW. We are expecting ~ 150kD and we are getting a smaller peak at that molecular weight and then two larger peaks at > 225kD. Isn't that too much of a difference to only be caused by differential glycosylation? When we analyze the same material by SDS-PAGE we get the expected MW values and generally the same banding pattern as the peaks, but the MW values are way off. This is why we suspect we may need a different MW standard for calibration.
Hi Shannon, Are you using the same MW standards for the SDS-PAGE and CE-SDS? How do the MW standards compare?
We are not using the same standards. The CE-SDS MW marker is proprietary from Beckman and I have been unable to find the protein identities. The standards compare in stated MW values with the high value for the CE marker at 225kDa and the high for the SDS-PAGE marker at 200kDa. Our mAb falls well below the 200kDa band on the gel and above the 225kDa peak in CE-SDS.
Ok, so can you run your SDS-PAGE MW markers on the CE-SDS and see how they line up with the MW CE-SDS markers from Beckman?
we tried running the SDS-PAGE MW markers on the CE instrument and were not able to get peaks.
You are not seeing any peaks when you run your SDS-PAGE MW markers on the CE-SDS??
That does not make sense.
You should contact the Beckman rep. to get an explanation on why SDS-PAGE markers are not showing up on their system.
I will have to try this again. I have not been running these experiments. Another scientist in the lab has been doing this work. I want to try mixing our mAb 50:50 with the ce-sds markers to see if the mAb still comes out after the 225kDa marker and also run them each alone. I can try the same with the sds-page markers. We are using an Agilent CE system.
Hi Shannon, I was looking at the Agilent CE system and it seems to me that applying high protein concentrations to this system can really screw up resolution.
Do you know if Agilent has an upper limit on the protein concentrations you can use on their system?
I will look into the protein concentration limit. We have performed some linearity studies with protein load and the results were fairly consistent for up to 5x our method load. The confusing thing is that our profiles look visually similar to what is expected based on Beckmann literature however the MWs are off. A collaborator of ours has also recently reported reducing CE-SDS results from a Beckmann system and they see higher than expected MW for the heavy chain which corresponds to our results. I am also having a difficult time finding reports of MW for mAbs in the literature. It seems that most papers are reporting relative quantities, but not MW. Are you familiar with any literature reporting accurate MW for mAbs?
When I want to find physical properties of antibodies, I usually look at literature from companies that do antibody isolation and characterization for a living. Like here:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-antibody-purification.pdf
According to company literatures that I have looked at, if your mouse monoclonal is a IgG subtype, there is no way that it is larger than 150kDa.
Unless you are loading an antibody-antigen complex onto the CE-SDS, I can't see how the MW of the Ab can be off by ~30-40%.
Yes, you are correct. This is a human mAb IgG with an expected MW of 147kDa. However, when calibrated with the beckmann ce-sds standards, it assigns a value of ~225kDa which is definitely too high. We are not loading an antibody-antigen complex. I can't see how it is off by so much either. This is why I would like to mix the mAb with the ce standards to see if the mAb peak still migrates after the 225kDa marker, even when run together in the same injection. I have not found much in the literature reporting an accurate MW for a mAb by CE-SDS.
225kDa is way to high for a human IgG, even with full glycosylation, and then some.
How good are the Agilent tech people? They might try to be weasels and tell you that your Ab is interacting with the standards.
Hi Shannon Renn-Bingham , I am facing the same issue. Were you able to solve this issue?
Hi Ritu Chandna, we did not solve the inaccurate MW assignment, but instead report %species (ie. % IgG or %HC + LC for mAbs) which is in-line with industry standards.
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