SEC-HPLC and aggregation QC
Size exclusion chromatography separates molecules by hydrodynamic size as they pass through a porous resin. Run on an HPLC with a UV detector, it is the standard measurement of how much of a protein preparation is monomer, how much is aggregate, and how much is fragment. For any material that will be stored, shipped or dosed, it is the number that gets watched.
Reading the chromatogram
A typical antibody trace has a main monomer peak, a high molecular weight shoulder or peak eluting earlier, and sometimes low molecular weight species eluting later. Percentages are computed from peak areas.
Two habits make the result trustworthy. Integrate consistently, with defined baseline and peak boundaries applied the same way across a panel or a stability series. And look at the trace, not only the table, because a shifting main peak or a growing tail is information that a monomer percentage hides.
The artifacts the method creates
SEC is not a passive observer. The column can change what it measures.
Column interaction. Proteins that interact with the stationary phase elute late, tail, or fail to elute at all. Aggregates are lost preferentially, so a molecule that sticks reports a falsely high monomer content. Adjusting the mobile phase, typically ionic strength or the addition of an organic modifier, usually fixes it, and the fix belongs in the method.
Dilution on column. Reversible self-association comes apart during the run, so weakly associating species can be invisible.
Sample stress. Filtering, diluting and sitting in an autosampler all change aggregate content. Keep the handling identical across samples that will be compared.
Orthogonal methods
Because of those artifacts, a stability claim that rests on SEC alone is weaker than it looks.
SEC with multi-angle light scattering gives absolute molar mass across the peak, which distinguishes a genuine dimer from a shape artifact.
Analytical ultracentrifugation measures in free solution with no stationary phase, so it catches what the column removes. Slow and low throughput, and the reference method when a number really matters.
Dynamic light scattering is fast, needs little material, and is very sensitive to large species. It is a good screening tool and a poor quantitative one.
Sub-visible particle counting covers the size range above what SEC sees, which is where a formulation problem first shows itself.
Thermal and colloidal stability
Aggregation QC usually travels with stability measurements: a thermal unfolding midpoint from differential scanning fluorimetry, an aggregation onset temperature, and sometimes an interaction parameter that describes whether molecules attract or repel each other at concentration. These predict which candidates will give trouble in formulation, and they are cheap enough to run across a panel.
Building a stability study worth having
Define the stress that matters for the program, which is usually time at a storage temperature, plus freeze-thaw cycles and agitation. Pull samples on a schedule, store them the same way, and run them on the same method with the same integration. Report the trend rather than a single time point, because the interesting result is the slope.
How we run it
We report monomer content with the method that produced it, check for column interaction before trusting a high number, pair SEC with an orthogonal method where a decision depends on it, and run stability as a trend rather than a single pull.