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

Tue Apr 8 09:20:21 PDT 2008

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

--
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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
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*******************************************************

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 

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