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<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>Allen, Ewa, and Arthur have already made some good
points about Bo's questions, but perhaps I can add and clarify a few
things.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>(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.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>(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). 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.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>(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.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>(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. </SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>(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).</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>(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. </SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Arial color=#0000ff size=2><SPAN
class=977332915-08042008>John</SPAN></FONT></DIV><BR>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left>
<HR tabIndex=-1>
<FONT face=Tahoma size=2><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <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></FONT><BR></DIV>
<DIV></DIV>
<BLOCKQUOTE><BR>Hi Allen<BR><BR>Good point, especially with regard to
antibodies.<BR><BR>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>Regards<BR><BR>Arthur<BR><BR><BR></BLOCKQUOTE>--<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:
www.nottingham.ac.uk/ncmh/business<BR>*******************************************************<BR><BR><BR>
<BLOCKQUOTE><BR><BR><TT>Hello Borries -<BR><BR>At 11:21 AM 4/8/2008, you
wrote:<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>1. Reversible self-association causes large increases in
viscosity <BR>(see recent work of Steve Shire and coworkers), which in turn
causes <BR>problems in administration of concentrated immunoglobulin via
<BR>injection through narrow bore needles. Different monoclonal
<BR>antibodies have significantly different tendencies to reversibly
<BR>self-associate at high concentration. Thus screening of candidate
<BR>engineered antibodies for tendency to reversibly self-associate at
<BR>high concentration is an essential part of the drug development
process.<BR><BR>2. Directly following administration there exists a bolus of
locally <BR>highly concentrated antibody at the site of administration which
<BR>requires time to dissipate. Reversibly formed aggregates therefore
<BR>exist with a significant lifetime. The physiological effects of such
<BR>aggregates are unknown, but it is possible that some oligomeric
<BR>species may be mistakenly identified as foreign proteins and <BR>therefore
break immune tolerance.<BR><BR>For both of these reasons the FDA wants
pharmaceutical companies to <BR>characterize formulations of monoclonal
antibodies with respect to <BR>both reversible and irreversible
self-association. The former <BR>requires measurement at high
concentration.<BR><BR>Allen
<BR><BR>_______________________________________________<BR>RASMB mailing
list<BR>RASMB@rasmb.bbri.org<BR>http://rasmb.bbri.org/mailman/listinfo/rasmb<BR></TT></BLOCKQUOTE><TT><BR></TT><BR>
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