<html>
<body>
<font size=2 color="#0000FF">Te marco en negrita una defensa de la
técnica de Allen por parte de John Philo que trabaja para la industria.
El resto de los comentarios tambien tiene su interes. Tambien puedes
incluirlo como aplicacion el el DEA.<br><br>
Carlos<br><br>
Allen, Ewa, and Arthur have already made some good points about Bo's
questions, but perhaps I can add and clarify a few things.<br>
</font> <br>
<font size=2 color="#0000FF">(1) Regarding SEC-MALLS, remember that the
MALLS does not help whatsoever in measuring the fractions of long-lived
(separable) species---it is the concentration detector (UV and/or RI)
that does this. The regulatory agencies are very aware that the column
can act as a filter, and also that typically the mobile phase is
different from the formulation buffer and this change in solvent
condition can change the distribution of aggregates. Probably 75% of the
SV work I do is therefore aimed at showing that SEC methods are actually
telling the truth (at least semi-quantitatively). What the MALLS adds is
the ability to actually identify the true MW of the minor species, i.e.
the stoichiometry of aggregates, which you really can't do based only on
a sedimentation coefficient. Knowing the true identity of the aggregates
is nice, but frankly it isn't essential because usually we can't really
say whether a trimer is worse than a dimer with regard to safety or
efficacy issues.<br>
</font> <br>
<font size=2 color="#0000FF">(2) Yes sometimes we see clear evidence of
rapidly-reversible association in a SEC-MALLS experiment (although often
it is not recognized as such by my clients even when they have acquired
such data themselves).<b> But if you want to measure Kd values for the
reversible association by LS then it is far better to get rid of the
column and use the approach pioneered by Allen Minton.<br>
</b></font> <br>
<font size=2 color="#0000FF">(3) The regulatory agencies would indeed
like to know the exact distribution of oligomers, both reversible and
irreversible, in the product vial, even for products at 100+ mg/mL.
Unfortunately they don't always understand that this may be technically
impossible with current technology and current theoretical understanding
of the strong non-ideality effects that may be present. They are indeed
worried about the possibility that the rapidly-reversible oligomers may
have biological effects during the period before the product is dispersed
in the body as Allen said (many of these high concentration products are
given subcutaneously). In my opinion there is little to no hard evidence
that the rapidly-reversible oligomers really cause trouble, but one
company has stated publically they think they did for one product so we
all have to deal with that issue.<br>
</font> <br>
<font size=2 color="#0000FF">(4) I agree with Arthur that to date most
biotech companies have not been terribly interested in reversible
association thermodynamics. This is probably going to change at least
somewhat due to (3) above. <br>
</font> <br>
<font size=2 color="#0000FF">(5) We tend to think of aggregates as either
irreversible or rapidly-reversible. Many RASMB readers may not realize
that it is not uncommon to see what I call "metastable"
aggregates which are reversible but dissociate very, very slowly (hours
to days). These seem to exist in a fair number of antibody preparations.
The slow dissociation allows you to separate and detect them at low
concentrations if the separation starts immediately after dilution (what
we call a "dilute and shoot" protocol).<br>
</font> <br>
<font size=2 color="#0000FF">(6) Although the biotech crowd would LIKE to
measure everything in the actual formulation buffer and at the
concentration in the vial, often this really isn't feasible. For these
high concentration antibodies at APL we mostly run SV at 0.5 mg/mL with
the "dilute and shoot" approach. And sometimes the formulation
contains things that cause significant interference (high levels of
sugar, detergents) and we may have to dilute into a different buffer to
get meaningful/reliable data. <br>
</font> <br>
<font size=2 color="#0000FF">John<br><br>
</font><hr>
<font face="Tahoma" size=2><b>From:</b> rasmb-bounces@rasmb.bbri.org
[<a href="mailto:rasmb-bounces@rasmb.bbri.org" eudora="autourl">
mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Arthur
Rowe<br>
<b>Sent:</b> Tuesday, April 08, 2008 8:24 AM<br>
<b>To:</b> Allen Minton; Borries Demeler<br>
<b>Cc:</b> rasmb@server1.bbri.org<br>
<b>Subject:</b> Re: [RASMB] non-ideality in velocity [was: interference
optics]<br><br>
<br>
</font>
<dl>
<dd>Hi Allen<br><br>
<dd>Good point, especially with regard to antibodies.<br><br>
<dd>However, I fear that the wide world of bio/pharma has yet to come to
terms with the need for both reversible and irreversible aggregation to
be characterised as universal practice.<br><br>
<dd>Regards<br><br>
<dd>Arthur<br><br>
<br>
</dl>--<br>
*******************************************************<br>
Arthur J Rowe<br>
Professor of Biomolecular Technology<br>
NCMH Business Centre<br>
University of Nottingham<br>
School of Biosciences<br>
Sutton Bonington<br>
Leicestershire LE12 5RD UK<br><br>
Tel: +44 (0)115 951 6156<br>
+44 (0)116
271 4502<br>
Fax: +44 (0)115 951 6157<br>
email: arthur.rowe@nottingham.ac.uk<br>
Web:
<a href="http://www.nottingham.ac.uk/ncmh/business" eudora="autourl">
www.nottingham.ac.uk/ncmh/business<br>
</a>*******************************************************<br><br>
<br><br>
<br>
<dl>
<dd><tt>Hello Borries -<br><br>
<dd>At 11:21 AM 4/8/2008, you wrote:<br><br>
<dd>>So my question is: Why are drug companies interested in the
reversible<br>
<dd>>kind, or is there another reason to measure at high
concentration?<br><br>
<dd>1. Reversible self-association causes large increases in viscosity
<br>
<dd>(see recent work of Steve Shire and coworkers), which in turn causes
<br>
<dd>problems in administration of concentrated immunoglobulin via <br>
<dd>injection through narrow bore needles. Different monoclonal
<br>
<dd>antibodies have significantly different tendencies to reversibly
<br>
<dd>self-associate at high concentration. Thus screening of
candidate <br>
<dd>engineered antibodies for tendency to reversibly self-associate at
<br>
<dd>high concentration is an essential part of the drug development
process.<br><br>
<dd>2. Directly following administration there exists a bolus of locally
<br>
<dd>highly concentrated antibody at the site of administration which
<br>
<dd>requires time to dissipate. Reversibly formed aggregates
therefore <br>
<dd>exist with a significant lifetime. The physiological effects of
such <br>
<dd>aggregates are unknown, but it is possible that some oligomeric <br>
<dd>species may be mistakenly identified as foreign proteins and <br>
<dd>therefore break immune tolerance.<br><br>
<dd>For both of these reasons the FDA wants pharmaceutical companies to
<br>
<dd>characterize formulations of monoclonal antibodies with respect to
<br>
<dd>both reversible and irreversible self-association. The
former <br>
<dd>requires measurement at high concentration.<br><br>
<dd>Allen <br><br>
<dd>_______________________________________________<br>
<dd>RASMB mailing list<br>
<dd>RASMB@rasmb.bbri.org<br>
<dd>
<a href="http://rasmb.bbri.org/mailman/listinfo/rasmb" eudora="autourl">
http://rasmb.bbri.org/mailman/listinfo/rasmb<br><br>
<br><br>
</a></tt>
</dl>This message has been checked for viruses but the contents of an
attachment may still contain software viruses, which could damage your
computer system: you are advised to perform your own checks. Email
communications with the University of Nottingham may be monitored as
permitted by UK legislation. <br>
_______________________________________________<br>
RASMB mailing list<br>
RASMB@rasmb.bbri.org<br>
<a href="http://rasmb.bbri.org/mailman/listinfo/rasmb" eudora="autourl">
http://rasmb.bbri.org/mailman/listinfo/rasmb<br>
</a><x-sigsep><p></x-sigsep>
Dr. Carlos Alfonso<br>
Servicio de Ultracentrifuga Analítica<br>
Centro de Investigaciones Biológicas<br>
<font face="Verdana">C/ Ramiro de Maeztu, 9. <br>
28040-MADRID<br>
Tlfno.- 91 </font>8373112<font face="Verdana"> ext.-4297 <br>
</font>Fax: 915360432.<br>
E-mail: uanalitica@cib.csic.es<br>
<b>NEW lab web page:
<a href="http://www.cib.csic.es/es/servicio.php?iddepartamento=5" eudora="autourl">
http://www.cib.csic.es/es/servicio.php?iddepartamento=5<br><br>
<br><br>
</a></b></body>
</html>