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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple style='word-wrap: break-word;-webkit-nbsp-mode: space;-webkit-line-break: after-white-space'><div class=WordSection1><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>John, first I am not very clear about what you mean by “failed to observe any evidence of non-ideal behavior”. Based on excluded volume alone one would expect the sedimentation coefficient of any globular protein to be at least ~4.5% less at 5 mg/mL than at infinite dilution (and more so for an asymmetric molecule like an antibody). So surely you must be seeing at least that effect, correct? If not, I would guess the decrease from non-ideality is being masked by weak rapid self-association, exactly as Jack said.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>If what you mean by “failed to observe any evidence of non-ideal behavior” is just that you can get a fairly good fit to something like a c(s) model without considering non-ideality, then yes at 5 mg/mL the fits might not look too bad. If all you are worried about is properties of major species, doing this may be okay, but as you go up in concentration at some point the non-ideality means the fitting model is fundamentally wrong and you will likely get false minor peaks. So a lot depends on exactly what is the purpose of the study.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>John<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b>From:</b> RASMB [mailto:rasmb-bounces@list.rasmb.org] <b>On Behalf Of </b>John J. Correia<br><b>Sent:</b> Wednesday, January 21, 2015 3:29 PM<br><b>To:</b> John Sumida; rasmb@rasmb.org<br><b>Subject:</b> Re: [RASMB] concentration range for non-ideal behavior<o:p></o:p></p></div></div><p class=MsoNormal><o:p> </o:p></p><div><p class=MsoNormal><span style='font-size:10.5pt;color:black'>John, The nonideality above 1 mg/ml rule depends upon the numerical contribution from a (1 + Ksc) term where c is on a weight scale. Ks is phenomenological and not easy to guess at. If Ks = 0.01 for example at 1 mg/ml the term starts to be significant. Thus if Ks is smaller than higher concentrations are required to nonideality effects. Since nonideality is either charge and/or excluded volume dependent, small molecules will not contribute much and uncharged molecules will not contribute. Thus working with small macromolecules at their pI will give ideal behavior over a wide range of concentrations. In addition mAb’s often self-associate albeit weakly thus potentially masking nonideality. <o:p></o:p></span></p></div><div><p class=MsoNormal><span style='font-size:10.5pt;color:black'><o:p> </o:p></span></p></div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='color:black'>From: </span></b><span style='color:black'>John Sumida <<a href="mailto:jpsumida@u.washington.edu">jpsumida@u.washington.edu</a>><br><b>Date: </b>Tuesday, January 20, 2015 at 8:00 PM<br><b>To: </b>"<a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a>" <<a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a>><br><b>Subject: </b>[RASMB] concentration range for non-ideal behavior<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='font-size:10.5pt;color:black'><o:p> </o:p></span></p></div><div><div><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'>Dear RASMB,</span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'> </span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'>My recollection is that concentrations in excess of 1.0 mg/ml should be avoided due to the possibility of non-ideal interactions that would complicate the analysis of SV and SE measurements. However in two recent cases (in one case a mAb was being studied, and in another a globular viral capsid protein), we exceeded this value by a factor of five and failed to observe any evidence of non-ideal behavior. In both cases, it was possible to fit both SV and SE data without assuming any non-ideal interactions when the experiment was performed in PBS or HEPES.</span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'> </span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'>I would be curious to hear if it is the experience of the community that non-ideality is commonly to be expected at concentrations >1.0mg/ml or whether the concentration cutoff for non-ideality is less of an actual cutoff being more a function of the particular properties of the molecule being studied.</span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'> </span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'>Best regards</span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'>John Sumida</span><span style='color:black'><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:black'>University of Washington</span><span style='color:black'><o:p></o:p></span></p></div></div><p class=MsoNormal><span style='font-size:10.5pt;color:black'>Individuals who have received this information in error or are not authorized to receive it must promptly return or dispose of the information and notify the sender. Those individuals are hereby notified that they are strictly prohibited from reviewing, forwarding, printing, copying, distributing or using this information in any way. <o:p></o:p></span></p></div></body></html>