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<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222">Hi Dr. Hayes!<o:p></o:p></span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">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.</span></p>
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mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">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.</span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">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
</span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;"> </span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">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.</span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">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.</span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">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.</span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;"><span style="font-size:12.0pt;font-family:"Arial","sans-serif";
mso-fareast-font-family:"Times New Roman";color:#222222"> </span><span style="color: rgb(34, 34, 34); font-family: Arial, sans-serif; font-size: 12pt;">Thanks
so much for your help!</span></p>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">- Ronald Toth</span></font></span></font></div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">Post Doctoral Researcher</span></font></span></font></div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">Macromolecule and Vaccine Stabilization Center</span></font></span></font></div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">University of Kansas</span></font></span></font></div>
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<div id="divRpF98486" style="direction: ltr;"><font face="Tahoma" size="2" color="#000000"><b>From:</b> David Hayes [drdavidbhayes@yahoo.com]<br>
<b>Sent:</b> Friday, October 04, 2013 12:04 PM<br>
<b>To:</b> RASMB<br>
<b>Cc:</b> Toth IV, Ronald T<br>
<b>Subject:</b> Re: [RASMB] Variability in association constants from sedimentation equilibrium data<br>
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<div><span>Hi Ronald,</span></div>
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<span>I did very few sedimentation equilibrium experiments, but I would say that I can relate to your current experience.</span></div>
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<span>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.</span></div>
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<span>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.</span></div>
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<span>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?)</span></div>
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<span>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).</span></div>
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<span>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.</span></div>
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<span>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.</span></div>
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<span>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.</span></div>
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<span>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.</span></div>
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<div style="color:rgb(0,0,0); font-size:16px; font-family:arial,helvetica,sans-serif; background-color:transparent; font-style:normal">
<span>David Hayes</span></div>
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<font size="2" face="Arial"><b><span style="font-weight:bold">From:</span></b> "Toth IV, Ronald T" <Toth.Ronald@ku.edu><br>
<b><span style="font-weight:bold">To:</span></b> "rasmb@rasmb.org" <rasmb@rasmb.org>
<br>
<b><span style="font-weight:bold">Sent:</span></b> Friday, October 4, 2013 12:23 PM<br>
<b><span style="font-weight:bold">Subject:</span></b> [RASMB] Variability in association constants from sedimentation equilibrium data<br>
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<div class="yiv1525038578MsoNormal">Hello members!</div>
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<div class="yiv1525038578MsoNormal">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?</div>
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<div class="yiv1525038578MsoNormal">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.</div>
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<div class="yiv1525038578MsoNormal">We had a relatively new user load the cells, could cross contamination between reference and sample combined with a mismatched meniscus cause this?</div>
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<div class="yiv1525038578MsoNormal">Thanks in advance for your feedback!</div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">- Ronald Toth</span></font></span></font></div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">Post Doctoral Researcher</span></font></span></font></div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">Macromolecule and Vaccine Stabilization Center</span></font></span></font></div>
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<div style="margin:0px"><font face="Times New Roman,serif" size="3"><span style="font-size:12pt"><font face="Tahoma,sans-serif" size="2"><span style="font-size:10pt">University of Kansas</span></font></span></font></div>
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