[RASMB] analysis of biotech samples [was: non-ideality in SV]

Wed Apr 9 08:06:35 PDT 2008

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.

Carlos

Allen, Ewa, and Arthur have already made some 
good points about Bo's questions, but perhaps I 
can add and clarify a few things.

(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.

(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.

(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.

(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.

(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).

(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.

John

----------
From: rasmb-bounces at rasmb.bbri.org 
[mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Arthur Rowe
Sent: Tuesday, April 08, 2008 8:24 AM
To: Allen Minton; Borries Demeler
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] non-ideality in velocity [was: interference optics]

Hi Allen

Good point, especially with regard to antibodies.

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.

Regards

Arthur

--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD   UK

Tel:        +44 (0)115 951 6156
            +44 (0)116 271 4502
Fax:        +44 (0)115 951 6157
email:      arthur.rowe at nottingham.ac.uk
Web:        www.nottingham.ac.uk/ncmh/business
*******************************************************

Hello Borries -

At 11:21 AM 4/8/2008, you wrote:

 >So my question is: Why are drug companies interested in the reversible
 >kind, or is there another reason to measure at high concentration?

1. Reversible self-association causes large increases in viscosity
(see recent work of Steve Shire and coworkers), which in turn causes
problems in administration of concentrated immunoglobulin via
injection through narrow bore needles.  Different monoclonal
antibodies have significantly different tendencies to reversibly
self-associate at high concentration.  Thus screening of candidate
engineered antibodies for tendency to reversibly self-associate at
high concentration is an essential part of the drug development process.

2. Directly following administration there exists a bolus of locally
highly concentrated antibody at the site of administration which
requires time to dissipate.  Reversibly formed aggregates therefore
exist with a significant lifetime.  The physiological effects of such
aggregates are unknown, but it is possible that some oligomeric
species may be mistakenly identified as foreign proteins and
therefore break immune tolerance.

For both of these reasons the FDA wants pharmaceutical companies to
characterize formulations of monoclonal antibodies with respect to
both reversible and irreversible self-association.   The former
requires measurement at high concentration.

Allen

_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org
http://rasmb.bbri.org/mailman/listinfo/rasmb

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.
_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org
http://rasmb.bbri.org/mailman/listinfo/rasmb

Dr. Carlos Alfonso
Servicio de Ultracentrifuga Analítica
Centro de Investigaciones Biológicas
C/ Ramiro de Maeztu, 9.
28040-MADRID
Tlfno.- 91 8373112 ext.-4297
Fax: 915360432.
E-mail: uanalitica at cib.csic.es
NEW lab web page: http://www.cib.csic.es/es/servicio.php?iddepartamento=5

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20080409/fc02c66d/attachment.html>