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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Hi John,<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">The accuracy of CFCA method depends on the actual protein and the assay setup. From our experience it is anywhere between 10 to 20%. One normally
runs at nM concentration of the samples and assumes that the mass transport is controlled by the diffusion of the monomeric species. You need to know D – measure, calculate, find in literature etc. Samples have to be homogeneous for the best performance
of the method. Variability of glycosylation level will add to the inaccuracy .<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Best regards,<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Natalia
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:black"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
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<div style="border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0cm 0cm 0cm">
<p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> John Sumida [mailto:jpsumida@u.washington.edu]
<br>
<b>Sent:</b> den 14 januari 2014 18:39<br>
<b>To:</b> 'Mattia Rocco'; Markova, Natalia (GE Healthcare); rasmb@rasmb.org<br>
<b>Subject:</b> RE: [RASMB] SPR query about CFCA applications<o:p></o:p></span></p>
</div>
</div>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">Natalia and Mattia,<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">Thank you for your responses. I gather that in general these values are not measured for the CFCA analysis and that perhaps there is not sufficient reason
to do so given the precision of the application? I wonder how variably glycosylated proteins would affect the accuracy of this technique?<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">Thank you both again for taking the time to respond.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">Best regards,<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">John Sumida, Ph.D.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:#1F497D"><a href="http://depts.washington.edu/cidb4bio/index.shtml">Analytical Biopharmacy Core Facility</a><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">University of Washington<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:#1F497D"><a href="http://www.moles.washington.edu/">Molecular Engineering & Sciences Institute, G22</a><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">3946 West Stevens Way NE<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue">Seattle WA 98195-1651<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Courier New";color:blue"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> Mattia Rocco [<a href="mailto:mattia.rocco@hsanmartino.it">mailto:mattia.rocco@hsanmartino.it</a>]
<br>
<b>Sent:</b> Tuesday, January 14, 2014 1:17 AM<br>
<b>To:</b> Markova, Natalia (GE Healthcare); John Sumida; <a href="mailto:rasmb@rasmb.org">
rasmb@rasmb.org</a><br>
<b>Subject:</b> Re: [RASMB] SPR query about CFCA applications<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal" style="margin-bottom:12.0pt"><span lang="EN-US"><br>
The f/f0 Natalia mentions is for almost spherical shape.... if you are dealing with an antibody, then the 1.5 value might be more appropriate (but I haven't done any calculations on it... ;-) )<br>
<br>
Best - Mattia<br>
<br>
At 08:13 AM 1/14/2014 +0000, Markova, Natalia (GE Healthcare) wrote:<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Content-Language: en-US<br>
Content-Type: multipart/alternative;<br>
boundary="_000_41C5780FD432C74DB68C588BE66E34F614EF6B76BUDURBPA08e2kad_"<br>
<br>
Hi John,<br>
<br>
<br>
<br>
We use as default f/f0 factor for globular proteins, which is 1.2 (not 1.5). However, user can freely enter any value for this factor in Web diffusion coefficient calculator (<a href="https://www.biacore.com/lifesciences/Application_Support/online_support/Diffusion_Coefficient_Calculator/index.html?section=lifesciences&realsection=lifesciences">https://www.biacore.com/lifesciences/Application_Support/online_support/Diffusion_Coefficient_Calculator/index.html?section=lifesciences&realsection=lifesciences</a>)<br>
<br>
You need to be registered on the Biacore website to access it.<br>
<br>
<br>
<br>
Best regards,<br>
<br>
<br>
<br>
Natalia<br>
<br>
<br>
<br>
<b><i>Natalia Markova (PhD) <br>
</i></b><br>
<i>GE Healthcare<br>
</i><br>
<i>R&D Life Sciences<br>
</i><br>
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</i><br>
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<i> <br>
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M +46 727 33 58 80<br>
<br>
E <a href="mailto:natalia.markova@ge.com">natalia.markova@ge.com</a><br>
<br>
<i> <br>
</i><br>
<a href="http://www.biacore.com/">www.biacore.com</a><br>
<a href="https://exchange1.ad.slu.se/owa/redir.aspx?C=61606803db87492ba25280057e5d208e&URL=http%3a%2f%2fwww.microcal.com%2f">www.microcal.com</a><br>
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<b>From:</b> <a href="mailto:rasmb-bounces@list.rasmb.org">rasmb-bounces@list.rasmb.org</a> [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>]
<b>On Behalf Of </b>John Sumida<br>
<b>Sent:</b> den 14 januari 2014 00:20<br>
<b>To:</b> <a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a><br>
<b>Subject:</b> [RASMB] SPR query about CFCA applications<br>
<br>
<br>
<br>
Dear RASMB,<br>
<br>
<br>
<br>
This question is in regards to an SPR application. We are currently investigating the use calibration free concentration analysis (CFCA) to monitor the stability of macromolecular preps over time in terms of their activity. Input parameters for this analysis
are ff0, viscosity, MW and D and we are using AUC to better determine both. Current applications seem to assume ff0=1.5, which may not be correct for monoclonal preps based on my reading of the literature.<br>
<br>
<br>
<br>
My query.<br>
<br>
1. Is it currently the common practice to measure these quantities and if so what is the preferred method to do so?<br>
<br>
<br>
<br>
Thank you all and Happy New Year,<br>
<br>
<br>
<br>
John Sumida, Ph.D.<br>
<br>
<a href="http://depts.washington.edu/cidb4bio/index.shtml">Analytical Biopharmacy Core Facility</a><br>
<br>
University of Washington<br>
<br>
<a href="http://www.moles.washington.edu/">Molecular Engineering & Sciences Institute, G22</a><br>
<br>
3946 West Stevens Way NE<br>
<br>
Seattle WA 98195-1653<br>
<br>
<br>
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