[RASMB] SPR query about CFCA applications

Markova, Natalia (GE Healthcare) Natalia.Markova at ge.com
Sun Jan 19 06:19:07 PST 2014


Hi John,

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 .

Best regards,
Natalia


From: John Sumida [mailto:jpsumida at u.washington.edu]
Sent: den 14 januari 2014 18:39
To: 'Mattia Rocco'; Markova, Natalia (GE Healthcare); rasmb at rasmb.org
Subject: RE: [RASMB] SPR query about CFCA applications

Natalia and Mattia,

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?

Thank you both again for taking the time to respond.


Best regards,
John Sumida, Ph.D.
Analytical Biopharmacy Core Facility<http://depts.washington.edu/cidb4bio/index.shtml>
University of Washington
Molecular Engineering & Sciences Institute, G22<http://www.moles.washington.edu/>
3946 West Stevens Way NE
Seattle WA 98195-1651



From: Mattia Rocco [mailto:mattia.rocco at hsanmartino.it]
Sent: Tuesday, January 14, 2014 1:17 AM
To: Markova, Natalia (GE Healthcare); John Sumida; rasmb at rasmb.org<mailto:rasmb at rasmb.org>
Subject: Re: [RASMB] SPR query about CFCA applications


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... ;-) )

Best - Mattia

At 08:13 AM 1/14/2014 +0000, Markova, Natalia (GE Healthcare) wrote:
Content-Language: en-US
Content-Type: multipart/alternative;
        boundary="_000_41C5780FD432C74DB68C588BE66E34F614EF6B76BUDURBPA08e2kad_"

Hi John,



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 (https://www.biacore.com/lifesciences/Application_Support/online_support/Diffusion_Coefficient_Calculator/index.html?section=lifesciences&realsection=lifesciences)

You need to be registered on the Biacore website to access it.



Best regards,



Natalia



Natalia Markova (PhD)

GE Healthcare

R&D Life Sciences

Rapsgatan 23

751 84 Uppsala

Sweden



M  +46 727 33 58 80

E   natalia.markova at ge.com<mailto:natalia.markova at ge.com>



www.biacore.com<http://www.biacore.com/>
www.microcal.com<https://exchange1.ad.slu.se/owa/redir.aspx?C=61606803db87492ba25280057e5d208e&URL=http%3a%2f%2fwww.microcal.com%2f>



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From: rasmb-bounces at list.rasmb.org<mailto:rasmb-bounces at list.rasmb.org> [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
Sent: den 14 januari 2014 00:20
To: rasmb at rasmb.org<mailto:rasmb at rasmb.org>
Subject: [RASMB] SPR query about CFCA applications



Dear RASMB,



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.



My query.

1.       Is it currently the common practice to measure these quantities and if so what is the preferred method to do so?



Thank you all and Happy New Year,



John Sumida, Ph.D.

Analytical Biopharmacy Core Facility<http://depts.washington.edu/cidb4bio/index.shtml>

University of Washington

Molecular Engineering & Sciences Institute, G22<http://www.moles.washington.edu/>

3946 West Stevens Way NE

Seattle WA 98195-1653


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