[RASMB] Variability in association constants from sedimentation equilibrium data

Fri Oct 4 15:00:06 PDT 2013

Hi Dr. Hayes!
 Thanks for your reply! The situation you described in your first paragraph is very close to what happened here, the other two rotor speeds are currently running.
 We are using absorbance optics because we don’t have 3 mm cells and the eventual goal is to collected high concentration data (60 mg/mL), which I think is too high for the interference optics, and I thought we could scan at a wavelength where we would catch the edge of the A280 peak, as there are no other peaks in the spectrum.
 We’ve seen a value reported for Ka in a paper on the order of 10^9. We are getting magnitudes of order lower, Ka’s anywhere between 1 and 9, but we are using a different buffer with a higher ionic strength, so I wouldn’t expect them to be comparable. We don’t have a known Ka for the mAb in this buffer. The reported value gives a Kd of ~0.001 mg/mL, which we’re pretty far away from, our lowest concentration is 0.5 mg/mL
 I think using under filled 2 sector cells is a great idea, more data would mean better fits, and the meniscus would be easier to match than using the top loaded cells. We have been using the match module in Heteroanalysis to test equilibrium.
 I asked about sedimentation velocity but was assured that it is pure, but I could definitely push harder on that point, if only to assess the reversibility of the association.
 It’s sounding more and more like we’re just missing data at the end of the sector, either from the sectors being too short or the complex being above 1 OD.
 Thanks so much for your help!

- Ronald Toth

Post Doctoral Researcher
Macromolecule and Vaccine Stabilization Center
University of Kansas
________________________________
From: David Hayes [drdavidbhayes at yahoo.com]
Sent: Friday, October 04, 2013 12:04 PM
To: RASMB
Cc: Toth IV, Ronald T
Subject: Re: [RASMB] Variability in association constants from sedimentation equilibrium data

Hi Ronald,

I did very few sedimentation equilibrium experiments, but I would say that I can relate to your current experience.
Every time I fit sed equilibrium data from the first speed (because the collaborator just could not wait a whole week for a peek at the answer) I was always completely wrong compared to the global fit to all three speeds.  If you are still fitting just one speed and getting strange answers, that would be what I would expect.

The limited concentration range of absorbance optics can also be a killer to getting a good data fit.  Do you know the expected kD?  Are you anywhere near the kD concentration?  Can you use interference optics?  You can also try 3 mm cells for higher concentrations and using A230 (depending on buffers) for low concentration.
I found the 6 sector cells too short in column height for really good sedimentation equilibrium data:  I just used an 8 hole rotor and lots of double sector cells filled with 150 - 200 uL.  And yes, it took longer to come to equilibrium, but the extra data was worth it.  (Also, did you use match or SEDFIT to test for equilibrium?)

Also, at most concentrations, the range of A280 linearity, which is about 1 OD really limits what you are actually fitting.  It could be that most of the dimer signal in your system is above 1 OD and you are not even really fitting it at all.  With a bit of work you can get SEDANAL to simulate any interacting system and also get output of the distribution of species.  With this information you can see how many data points that you are actually including in the fit are significantly incorporating signal from dimer (this calculation depends on assuming a kD, so actually you will get a set of answers from a set of guessed kD values).

Sed equilibrium with absorbance optics works better than that with interference optics because you don't have to float the baseline.  But you say that some sectors are mismatched, so maybe you are floating the baseline.  Bad baselines give you bad fits.

I also found it useful to "screen" each channel by doing a fit to a single species without floating the baseline at all as an estimate of that sector's average sigma.  You should see a trend in the sigma versus concentration for a self associating system.  Mis-filled or noisy channels then stick out without making assumptions.

So in conclusion, my personal rule is 3 speeds, 3 concentrations, global fit, or don't tell anyone anything.  I started using 4-5 speeds and 4-5 concentrations after getting burned by having less data.

Also, have you done sed velocity?  How pure is this stuff?  How much irreversible dimer and larger species is in it?  If the kD is near 0.5 mg/ml, then you could get the kD from a concentration series sed velocity data set.  If the kD is far from 0.5 mg/ml, you won't get any good sed equilibrium or sed velocity data at 280 nm.

David Hayes

________________________________
From: "Toth IV, Ronald T" <Toth.Ronald at ku.edu>
To: "rasmb at rasmb.org" <rasmb at rasmb.org>
Sent: Friday, October 4, 2013 12:23 PM
Subject: [RASMB] Variability in association constants from sedimentation equilibrium data

Hello members!

We have a project where we are trying to extract association constants from sedimentation equilibrium data of a self-associating mAb. We are noticing a lot of variability in the association constants fit to between concentrations and even between replicates of the same concentration. My question is, is this expected? If not, what are some possible reasons it could be happening and what can I do to resolve it?

I’ll give more information in case it is relevant. We are using Beckman 6-sector cells and absorbance optics at 280 nm. Our initial guess as to rotor speed was a little high and gave sigma’s between 4 and 5. In some sectors there was a mismatch between sample and reference, but nothing in the buffer absorbs at 280 nm. In the fit we are using Heteroanalysis with a monomer-nmer model and constraining the molecular weight and stoichiometry.

We had a relatively new user load the cells, could cross contamination between reference and sample combined with a mismatched meniscus cause this?

Thanks in advance for your feedback!

- Ronald Toth

Post Doctoral Researcher
Macromolecule and Vaccine Stabilization Center
University of Kansas

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