Comparison size-exclusion chromatography (SEC) / Analytical Ultracentrifugation (AUC)

Currently the most widely used method for the characterisation of aggregation is size-exclusion chromatography (SEC). It is easy to use, relatively fast and requires only low amounts of sample for a sensitive and reproducible analysis of biopharmaceuticals. In combination with a “cheap” instrumentation and the possibility to automate most of the analysis, size-exclusion chromatography has become the industry’s workhorse for the analysis of biopharmaceuticals. These advantages come at a cost, however. Size-exclusion chromatography generally requires a change in solvent for analyis and considerable dilution of the sample occurs between the points of sample injection and detection. Both factors can potentially alter the amount and distribution of aggregates initially contained in the sample. This is especially true for weakly or reversibly aggregated material. An extensive interface exists between the SEC-column matrix and the mobile phase, posing a high risk of non-specific interactions. Finally, the separation range of SEC-matrices may not be suitable to accommodate all sizes present in a given sample and hence limit the information obtainable about the true size-distribution. All these factors contribute to the considerable risk of size-exclusion chromatography for false positive and false negative signals.

Analytical Ultracentrifugation (AUC) is in many respects the opposite of SEC. It is a non-trivial method that hasn’t found widespread use, though it has been on the rise in recent years, due to several unique advantages. It requires “expensive” instrumentation as well as specialized and highly trained staff to run the instrument and obtain good quality data. Analytical Ultracentrifugation is a comparatively slow method, in addition requiring extensive data analysis by experienced personnel. Due to these factors, it cannot be automated and is certainly “low-throughput”. On the other hand, no change in solvent is required for an analyis by Analytical Ultracentrifugation and minimal dilution occurs during the analysis. Therefore, Analytical Ultracentrifugation is not perturbing the initial distribution of aggregates. No matrix exists in Analytical Ultracentrifugation, the separation occurs by centrifugal force only and in the native solvent. The interface between the cuvette and the solution is minimal and physically inert materials are being used, further minimizing the risk of altering the sample. The size range accesible in a single AUC-experiment easily covers four orders of magnitude on the molar mass scale. This ensures that the native size-distribution of the sample is correctly reflected in the analysis. Taken together, these attributes make Analytical Ultracentrifugation the ideal orthogonal method to validate and confirm a given SEC-result. Moreover, AUC determines several other parameters not accessible by SEC.

Here’s a comparison between the two methods, where the number of pluses indicates the extent of the effect:

  SEC AUC
Change of solvent +++ -
Dilution +++ +
Matrix interaction +++ -
Adsorption to interfaces +++ +
Physical filtration of large aggregates +++ -
Disruption of weak aggregates +++ -
Time of measurement Fast (~ 15 mins) Slow (~ 4 hours)
Automation +++ -

 

In a recent comparative study, Gabrielson et al. have compared the performance of SEC, asymmetrical flow field flow fractionation (AF4) and SEC in detecting aggregates of acid-stressed and unstressed humanized monoclonal antibodies (mAbs). They found that SEC significantly underestimated the aggregate content of the stressed samples in comparison to Analytical Ultracentrifugation and AF4. Moreover, SEC failed to detect higher molecular weight aggregates apparent in AUC and AF4. These results make a strong point to use Analytical Ultracentrifugation as an orthogonal method for developing and validating SEC-protocols.

Reference:

Gabrielson JP, Brader ML, Pekar AH, Mathis KB, Winter G, Carpenter JF & Randolph TW (2007) Quantitation of Aggregate Levels in a Recombinant Humanized Monoclonal Antibody Formulation by Size-Exclusion Chromatography, Asymmetrical Flow Field Flow Fractionation, and Sedimentation Velocity. J Pharm Sci 96(2):268-279.