James,
Turbidity at 340 nm is one way to follow relative amounts of aggregate
formation, but it is not very quantitative. For example, two samples could
have the same turbidity but one could have a large amount of small
aggregates and the other a small amount of large ones.
The most common method for measuring soluble aggregates is size-exclusion
chromatography. One major drawback of SEC is that seldom can it be run using
the formulation buffer as the elution buffer, and the high ionic strength
elution buffers that usually must be employed may actually induce new
aggregates. The second major issue is that the aggregate species, and
especially large ones, are usually MUCH more sticky than the native
conformation, and thus may never elute from the column. Trying to adjust the
elution buffer to get good recovery of all species only aggravates the first
problem. Third, the dilution on the column may cause noncovalent aggregates
to dissociate. Thus overall it is hard to be certain that what you are
measuring truly reflects what was in the sample before the measurement.
Sedimentation velocity is an excellent method for quantitating aggregates,
and it can generally be done directly in the formulation buffer. With the
latest methods it is possible to detect species present at only a few tenths
of a percent. The throughput is low, so it is not generally used routinely,
but rather as an absolute method to validate or back up other approaches.
This is something we do a lot of on a contract basis at Alliance Protein
Labs, and there are examples on our web site, www.ap-lab.com
Dynamic light scattering is very good at detecting aggregates, especially
very small amounts of large ones (which it may detect at levels of only
0.01% by weight). It is often a good approach to detecting the smaller
precursors to the visible "snow" or "floaters" that plague protein
formulations. While it can detect the aggregates, it is usually only
semi-quantitative about their relative amount. Nonetheless, it is usually a
good method for tracking down the source of the stress causing the
aggregation, comparing one formulation to another, etc. This is another
method we can do on a contract basis.
For smaller aggregates (dimer-decamer) adding on-line classical light
scattering to SEC allows you to unambiguously identify the mass of the
aggregate. We have examples of this application on the web site also. Of
course, this method inherits the drawbacks of SEC mentioned above.
'Hope this helps,
John Philo
Alliance Protein Laboratories
www.ap-lab.com
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of James_Freedy@hgsi.com
Sent: Monday, February 12, 2001 12:51 PM
To: Recipients of ABRF List
Subject: aggregates
Question, anyone have experience with determining aggregate formation in
protein solutions? I am in need of a good method/instrument to quantitate
both soluble and insoluble aggregates. This need is based on the fact that
the particular protein solution becomes cloudy with time and under stressed
conditions and eventually forms a white precipitate. I have not actually
seen this yet, but am basing this on other's observations.
Anyone have any luck with light scatter measured at 340nm?
I am just getting started and could us any suggestions.
Thanks to all, in advance.
James G. Freedy
Senior Analyst
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