Two excellent methods for analyzing aggregates in monoclonals (and ones
which we have employed for our pharmaceutical clients) are 1) on-line
classical light scattering used in conjunction with size-exclusion
chromatography and 2) sedimentation velocity.
While conventional SEC can see the aggregates as early-eluting peaks or
shoulders, you can not reliably determine their molecular mass based on
their elution position. (This is particularly true for asymmetric molecules
like antibodies where the hydrodynamic size is strongly dependent on whether
the aggregates form end-to-end or side-to-side, and we have seen cases where
antibody aggregates were miss-identified based on their elution position).
The light scattering detection gives the true mass independent of elution
position. The scattering detection also gives you greater sensitivity for
detecting small amounts of very large aggregates than you get with
absorbance detection alone.
A problem with SEC approaches (with or without light scattering detection)
is that the column may filter out the aggregates you are trying to detect
(the aggregates are generally more sticky than the native state).
Sedimentation velocity has a great advantage over SEC in that there is no
potential loss of material to the column matrix, and you can work in buffers
at or below physiological ionic strength. (In SEC you usually work at very
high ionic strength to try to minimize sticking to the column, but those
high ionic strength conditions may alter the distribution you are trying to
measure). Sedimentation velocity can detect as little as 1% aggregate.
Lastly, I should mention that sometimes the aggregates are disulfide-linked,
and thus can be simply detected by non-reducing SDS gels.
'Hope this helps,
John Philo
Alliance Protein Laboratories
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Michael Moore
Sent: Thursday, April 08, 1999 1:26 PM
To: Recipients of ABRF List
Subject: antibody analysis
Does anyone have a suggestion for measuring the amount of agragated vs.
non-agragated monoclonal antibody. The MW is approximately 150kD, and the
agragates are most likely greater than dimers. Thanks in advance
Michael