Interview with Hillel Brandes (Sigma Aldrich) on advances in bioseparation of large molecules

Hillel Brandes completed his doctorate in biochemistry from the University of Missouri-Columbia in 1990. He then held positions as a postdoctoral associate and research assistant professor in the Protein Chemistry & Engineering Program at Oak Ridge National Lab in conjunction with the University of Tennessee. In 1997 Hillel joined the bioseparations group at Supelco and participated in the development of the Discovery^®, Discovery BIO and Ascentis® HPLC column lines. Currently Dr Brandes is a Principal Applications Chemist in Supelco’s Analytical Research Services group, where he focuses on applying the benefits of Fused-Core^® and traditional high-performance columns for biopolymer separations with special emphasis on biotherapeutics.

Could you tell Bioanalysis Zone a little about your current role at Supelco?

My main role at Supelco is HPLC application support for our bioseparations product lines. This involves demonstrating the utility of our modern HPLC columns in addressing the analytical needs of our customers in the biotech, biopharmaceutical and academic market segments.

At what stage in your career did you first become involved in the development of HPLC columns and what first attracted your attention to this area?

I first became involved with development of HPLC columns shortly after arriving at Supelco. Prior to that, I had developed a working expertise in bioseparations of peptides and proteins. This was done in support of basic research to understand the activity and regulation of enzymes from a structure-function perspective. I always enjoyed the challenge of chromatographic method development from a pragmatic  standpoint of maximizing resolution, throughput, and taking advantage of selectivity. Part of effective method development, is of course, appropriate choice of the stationary phase. A logical extension of this was an interest in column chemistries that give the user unique tools to address their separation needs.

In the past, core-shell columns have been used for small molecules. You have recently developed core-shell columns that are suitable for peptides and other large molecules. What are the challenges you have faced when adapting this technology for use with large molecules?

Core-shell columns for large molecule analysis have only come on the market in the last few years. That is, core-shell columns with pore sizes between 150 and 400 Å. Prior to this, core-shell columns were only available for small to intermediate-sized molecules. The major challenge in adapted the technology of core-shell type particles for large molecule separations, has been in identifying best options for pore size, shell thickness and particle size. The topic of optimum pore size for the separation of biological macromolecules, or fragments thereof, is not new. Of course, selection of a pore size(s) will be a compromise and no one pore size is optimal for all samples. Appropriate shell thickness will be affected by the mechanism of interaction of the analyte with the stationary phase, as well as diffusion rates between the bulk solvent and the stationary phase. Optimum particle size will be determined in part, by a compromise between a desire for maximizing resolution (i.e. smaller particles), pore size and optimizing flow (large analytes have relatively low diffusion rates).

During purification, proteins are often in the presence of surfactants to inhibit protein aggregation or adsorption. What methods can be used to measure the level of aggregation?

There are a variety of physicochemical methods for measuring aggregates, depending on the size of the aggregate (asymmetric field flow fractionation, optical microscopy, resonant mass measurement, Coulter principle). But as for chromatographic methods, traditionally, size-exclusion has been the approach taken, though more recently some have demonstrated the application of hydrophobic interaction chromatography for antibody aggregate analysis.

Could you tell us a bit about challenges to method development for applications involving protein and biotherapeutic analyses?

Traditionally, reversed phase separations of peptides and proteins have been with water-acetonitrile mobile phase systems with dilute TFA to affect pH and ion-pair, and performed at moderate temperatures with UV detection. Today, LC-MS is becoming more and more popular, and so this may dictate choice of alternate pH control or choice of buffers. Additionally, biotherapeutics may represent cases of unusual analyte hydrophobicity, and so generic methods may not always be satisfactory. The chromatographer may need to consider alternate mobile modifiers, additives and/or operate at elevated temperature.

What are you excited about working on over the next year?

Elucidating and developing more effective chromatographic strategies for biotherapeutic analysis and characterization.