<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Borries,<div> Your point regarding reversible aggregation is well taken. However, some of the concerns that companies have is when given as a bolus IV what is the impact on association due to molecular crowding? This situation may even be worse when using the SC route of administration, and coupled with the fact that immune responses are greater when given by this route there is concern regarding impact of a crowded environment on the association of therapeutic proteins. In addition, even before administration is the physical properties of the drug in the vial. We have observed high viscosities of drug formulation at >100 mg/mL which appear to be related to protein self association (Liu et al., J. Pharm Sci., 94,1928 (2005) and Kanai et al. J. Pharm. Sci. in press, available on-line. These high viscosities can be a problem when trying to deliver the drug by injection.</div><div> Steve<br><div><div>On Apr 8, 2008, at 8:21 AM, Borries Demeler wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite"><blockquote type="cite"><br></blockquote><blockquote type="cite">John -<br></blockquote><blockquote type="cite"><br></blockquote><blockquote type="cite">Yes, I take your point. I think we both know the 'biotech crowd', and the<br></blockquote><blockquote type="cite">desire which they can often have for results obtained under 'formulation<br></blockquote><blockquote type="cite">conditions'. But of course there may be a real problem involved in<br></blockquote><blockquote type="cite">correcting for Johnston-Ogston effects.<br></blockquote><br>John and Arthur,<br><br>Aside from the points about J-O effect and other non-ideality effects, I<br>think that the premise of the 'biotech crowd' of evaluating formulations<br>exclusively at high concentration should be re-evaluated. Formulation<br>conditions often require high concentrations of the solute, and drug<br>companies are interested in the percentage of undesirable aggregate. <br><br>I think it is not unreasonable to separate the "aggregates" into<br>2 kinds: reversible oligomerization (not necessarily undesirable)<br>and irreversible aggregation. The amount of reversible aggregate is<br>clearly related to concentration, the higher the concentration, the<br>more you have. Once the formulation is injected, the solute is diluted<br>in the blood stream and presumably at a concentration resembling a low<br>concentration AUC experiment (or lower), so why worry about it? If one<br>really were interested in the Kd, and if it were high, an SE experiment<br>at higher concentration may be more appropriate to determine the amount<br>of reversible oligomerization, without having to worry about pitfalls<br>of SV at high concentration.<br><br>This leaves the irreversible aggregates, which really are undesirable.<br>Even upon dilution, the percent of irreversible aggregation will not<br>change, because, by definition, those aggregates are irreversible, and<br>therefore any method measuring the formulation at low concentration should<br>provide the correct percentage, with the added benefit of not having to<br>worry about J-O effects, boundary sharpening, and Weiner skewing etc.<br><br>So my question is: Why are drug companies interested in the reversible<br>kind, or is there another reason to measure at high concentration?<br><br>Incidentally, it has been my experience that SEC-MALS is not so great<br>at detecting large irreversible aggregates, because they can get stuck in<br>the SEC column and you never see them in the MALS detector. However,<br>the reversible oligomerization may be detected fine by MALS .<br><br>Comments?<br><br>-Borries<br>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><br>http://rasmb.bbri.org/mailman/listinfo/rasmb<br></blockquote></div><br><div apple-content-edited="true"> <span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><div style="word-wrap: break-word; -khtml-nbsp-mode: space; -khtml-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Geneva" size="3" style="font: 12.0px Geneva; font-family: Geneva; "><span class="Apple-style-span" style="font-family: Geneva; "><span class="Apple-style-span" style="font-family: Geneva; "><span class="Apple-style-span" style="font-family: Geneva; ">Steven J. 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