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</o:shapelayout></xml><![endif]--></head><body lang=EN-GB link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:11.0pt;color:black'>John: because of the tumbling motion of the macromolecule under shear, the viscosity increment is more related to the rotational friction coefficient(s) </span><span style='font-size:11.0pt;font-family:Symbol;color:black'>r/ro</span><span style='font-size:11.0pt;color:black'>– from fluorescence depolarization anisotropy decay, electric birefringence decay etc. than the translational frictional ratio f/fo. Like the viscosity increment, the rotational frictional coefficients are also very sensitive to conformation. You can see the difference in sensitivities between these and the translational f/fo in Tanford’s 1961 book for simple ellipsoids of revolution, and between related parameters that don’t require assumptions over hydration in Harding & Rowe, Int. J. Biol. Macromol., 4, 160, 1982.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>For globular particles of approximately the same degrees of hydration – whether they be proteins or large viruses - the intrinsic viscosity is remarkably insensitive to molar mass (with ovalbumin and tomato bushy stunt virus having approximately the same values ~3-4 ml/g) . As soon as you depart from sphericity then it becomes also very sensitive to molar mass.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>So for say aggregated antibodies they wouldn’t necessarily show any real difference in intrinsic viscosity - and hence absolute viscosity for the same concentration. Absolute viscosity would depend of course strongly on concentration. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>Hope this helps!<br>Steve<o:p></o:p></span></p><div><p class=MsoNormal><i><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:yellow'><br><br></span></i><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:yellow'><o:p></o:p></span></p></div><div><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> 04 June 2015 00:40<br><b>To:</b> 'Razinkov, Vladimir'; 'Stephen Harding'; 'David Hayes'; 'RASMB'<br><b>Subject:</b> RE: [RASMB] Viscometer technology<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>Dear all, <o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>thank you for your explanations. <o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>Regarding the conformational effects on sedimentation, for a heterogeneous solution, since viscosity (dynamic viscosity) is directly related to friction, what is the relative agreement between ff0 that one might fit and the value one might obtain from a viscometric measurement of the same heterogeneous solution – I’m assuming that measuring ff0 is a possibility with this technology. <o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>I suppose there would be some interplay between the effect of the aggregate content and the presence of proteins with different conformation; and, I suppose that smaller faster diffusing molecules would show larger differences in viscosity than larger more slowly diffusing molecules?<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>Best regards<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'>University of Washington.<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><div><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"'> Razinkov, Vladimir [<a href="mailto:razinkov@amgen.com">mailto:razinkov@amgen.com</a>] <br><b>Sent:</b> Wednesday, June 03, 2015 2:13 PM<br><b>To:</b> Stephen Harding; John Sumida; 'David Hayes'; 'RASMB'<br><b>Subject:</b> RE: [RASMB] Viscometer technology<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:"Calibri","sans-serif";color:black'>For viscosity measurements of small volume protein solutions we use Wyatt DynaPro plate reader and 384-well plate. It takes ~ 35 uL of protein sample. Diffusion coefficient of added into solution beads with known radius is measured and converted to viscosity. See our original article: </span><b><span lang=EN-US style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black;background:white'>He, Feng, et al. "High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions."<span class=apple-converted-space> </span><i>Analytical biochemistry</i>399.1 (2010): 141-143</span></b><span lang=EN-US style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black;background:white'>. It is good tool for quick selection of viscous candidates.<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black;background:white'>Protein-bead interactions and subsequent aggregation can be a problem. But, based on our experience, it is rare for antibodies. <o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black;background:white'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;font-family:"Arial","sans-serif";color:black;background:white'>Vladimir Razinkov<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;font-family:"Arial","sans-serif";color:#222222;background:white'><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'><o:p> </o:p></span></p><div><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"'> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>Stephen Harding<br><b>Sent:</b> Wednesday, June 03, 2015 1:45 PM<br><b>To:</b> John Sumida; 'David Hayes'; 'RASMB'<br><b>Subject:</b> Re: [RASMB] Viscometer technology<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 style='font-size:11.0pt;color:black'>John: interest in protein intrinsic viscosity is that it is a far more sensitive function of conformation in solution compared with the sedimentation coefficient. In addition, interest in absolute viscosity is important in for example monoclonal antibody based formulations – high viscosity is a problem in terms of administration.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>For conformation work, problem in the past has been that of sample heterogeneity – sedimentation velocity could resolve away aggregates etc, viscometry couldn’t – not until the advent of the differential pressure viscometers (measuring relative viscosity by pressure tranducers rather than photosensors) – which could be linked onto SEC columns and concentration detectors. These instruments (Wyatt Viscostar or the Malvern Viscotek) are so sensitive that they can measure relative viscosity at concentrations low enough so that a concentration extrapolation of the reduced viscosity to zero concentration c=0 are not necessary (there are other tricks as well!).<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>There have been difficulties largely arising from corrosion of the transducers after being exposed to salt ions in aqueous solution but they seem to have got round that now by appropriate coating - indeed its nice instrumentation.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>David – yes the rolling ball viscometer (Anton Paar) although its been good for looking at polysaccharide solutions – the ability to vary the angle of flow changes the shear rate so you can monitor for non-Newtonian behaviour as with Cone and Plate (but much lower volumes) is really useful….. but bad for protein solution work. That was the view 5 years ago at the great BITC meeting in Galveston – and is still the same. The Anton-Paar team were really helpful when we approached them about silanization and this improved things but not really enough. For polysaccharides yes, proteins no. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>For intrinsic viscosity measurement on proteins we would probably still go for the standard Ostwald U-tube capillary (1st choice) or if polydispersity is suspected the Viscostar (2<sup>nd</sup> choice). For polysaccharide/ glycoconjugate work, the Viscostar would be 1<sup>st</sup> choice.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>The new Malvern Viscosizer is based on a different principle altogether. Malvern for some time have been trying to see if dynamic light scattering on particles of known size can be used as a measure of the viscosity of a medium from the relation of the diffusion coefficient with viscosity, and the Taylor dispersion based instrument I guess is a development from that so it would be interesting to see how it goes. The problems of protein adhesion sounds ominously familiar! It looks to me anyway as if its intended for absolute viscosity measurement rather than being accurate enough for intrinsic viscosity. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>All best, <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;color:black'>Steve<o:p></o:p></span></p><div><p class=MsoNormal><i><span lang=DE-CH style='font-family:"Verdana","sans-serif";color:red'>________________________</span></i><span lang=DE-CH style='font-family:"Verdana","sans-serif";color:red'><o:p></o:p></span></p><p class=MsoNormal><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>Steve Harding DSc (Oxon), RFOO (Norway)<o:p></o:p></span></p><p class=MsoNormal><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>Professor of Applied Biochemistry,<o:p></o:p></span></p><p class=MsoNormal><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>University of Nottingham,<o:p></o:p></span></p><p class=MsoNormal><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>School of Biosciences,<o:p></o:p></span></p><p class=MsoNormal><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>Sutton Bonington LE12 5RD, England<o:p></o:p></span></p><p class=MsoNormal><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>(44) (0) 78110 90635<o:p></o:p></span></p><p class=MsoNormal style='margin-bottom:12.0pt'><u><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'><a href="http://www.nottingham.ac.uk/Biosciences/People/steve.harding"><span style='color:black'>www.nottingham.ac.uk/Biosciences/People/steve.harding</span></a><o:p></o:p></span></u></p><p class=MsoNormal><span style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>National Centre for Macromolecular Hydrodynamics (NCMH):<b><o:p></o:p></b></span></p><p class=MsoNormal><span style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'><a href="http://www.nottingham.ac.uk/ncmh"><span style='color:black'>www.nottingham.ac.uk/ncmh</span></a><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>Genetic Survey of North West England:<b><br></b><a href="http://www.nottingham.ac.uk/-sczsteve/survey.htm"><span style='color:black'>www.nottingham.ac.uk/-sczsteve/survey.htm</span></a><o:p></o:p></span></p><p class=MsoNormal><span style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:black'>(BBSRC Watson-Crick Anniversary Award)<o:p></o:p></span></p><p class=MsoNormal style='margin-bottom:12.0pt'><i><span lang=DE-CH style='font-family:"Verdana","sans-serif";color:yellow'>________________________</span></i><span lang=DE-CH style='font-size:6.0pt;font-family:"Verdana","sans-serif";color:yellow'><o:p></o:p></span></p></div><p class=MsoNormal><span style='font-size:11.0pt;color:black'><o:p> </o:p></span></p><div><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"'> RASMB [<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> 03 June 2015 17:32<br><b>To:</b> 'David Hayes'; 'RASMB'<br><b>Subject:</b> Re: [RASMB] Viscometer technology<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span lang=EN-US style='font-size:11.0pt;font-family:"Courier New";color:blue'>Dear David,<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'>Your post piqued my curiosity. Please forgive my ignorance on the topic, but why is protein viscosity something one would be interested in? And for those who make these types of measurements, what is the variability in protein viscosity?<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 all for your comments and responses, now and in the past. This bulletin board has been a great learning tool.<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<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:blue'><o:p> </o:p></span></p><div><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"'> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>David Hayes<br><b>Sent:</b> Wednesday, June 03, 2015 5:23 AM<br><b>To:</b> RASMB<br><b>Subject:</b> [RASMB] Viscometer technology<o:p></o:p></span></p></div></div><p class=MsoNormal><span lang=EN-US><o:p> </o:p></span></p><div><div><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>Hi all (especially hoping Steve Harding is active),<o:p></o:p></span></p></div><div><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'> <o:p></o:p></span></p></div><div><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>I recently saw the Malvern viscosizer. It is a capillary viscometer and taylor dispersion instrument. (Taylor dispersion looks at the diffusion pattern of a small plug of sample in a capillary). Because of the small volumes used, it could be useful for screening early limited supply proteins for viscosity problems.<o:p></o:p></span></p></div><div><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'> <o:p></o:p></span></p></div><div><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>Are there any general thoughts on the technology?<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3436"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'> <o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3437"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>I remember Steve Harding telling us at BITC that you never use rolling ball viscometers on proteins: the proteins stick to the ball.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3438"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>Recently we found out here that the regular Anton Paar cone and plate rheometers sometimes need PS-80 in protein samples or the air water interface around the outside edge can predominate the viscosity measurements.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3439"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'> <o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3440"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>I did ask whether most samples just stick to the capillary and disappear or run anomalously, but the answer was they had a super coating so most things won’t stick. This coating makes the capillary impossible to clean (so you buy more capillaries from Malvern). And that the taylor dispersion fitting will tell you if and approximately how much the sample interacts with the capillary walls.<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3441"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'> <o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3442"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>Kind Regards,<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3443"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'> <o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3457"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'>David Hayes<o:p></o:p></span></p></div><div id="yui_3_16_0_1_1433333972801_3394"><p class=MsoNormal style='background:white'><span lang=EN-US style='font-family:"Helvetica","sans-serif";color:black'><o:p> </o:p></span></p></div></div><pre><o:p> </o:p></pre><pre><o:p> </o:p></pre><pre><o:p> </o:p></pre><pre>This message and any attachment are intended solely for the addressee<o:p></o:p></pre><pre>and may contain confidential information. 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