Biochromatography: The New Frontier in Chromatography?

Incognito has witnessed the rise of biochromatography and it looks exciting!

Photo Credit: Studio-Pro/Getty Images

Back in February 2009 I described what I saw as the inexorable rise of the biochromatographer.¹ Over six years later, I’m glad to report that I attended a meeting of such folks in May 2015 and many of my original predictions have come true.

What amazed and delighted me was that I saw a level of enthusiasm, debate, and forward thinking that I have not seen in the “small-molecule” world for many years. I wanted to pass on some of these glad tidings, not to gloat, but as an attempt to highlight the current debate within the world of large-molecule analysis and to stimulate thought amongst those of us working in the small molecule world, who might benefit from a rethink of our current perception that we have seen all there is to see. Where the only thing to look forward to is the inexorable decay into an industrialized future where pressing button A results in data emerging from chromatography data system B, which is then emailed to colleagues to “do something with”.

This was a meeting involving industrial biochromatographers, but let me further elucidate. Fellow delegates included those who had been analyzing proteins and peptides for many years and who vendors were now telling (perhaps because there is a buck to be made in “new technology”?) that they had been doing things incorrectly for many years and they would be shown the “new way” - much to their amusement obviously! Then there were the small molecule folks - you could spot them by the bewildered looks on their faces - learning how to analyze the products that formed the basis of their company’s future. No pressure there then…

While I don’t want this month’s column to be a glorified meeting report, I do want to highlight some of the key analytical challenges, debates, and new developments, which will stimulate thought no matter what the scale on your mass axis or how many charges your analyte molecule is capable of carrying.

It appears that the analysis of peptides and proteins regularly requires the use of several different modes of chromatography, all working in unison to help to characterize and quantify the biological entity. Reversed-phase high performance liquid chromatography (HPLC) is typically used to analyze smaller intact analytes (using wide-pore stationary phases); or the digested products of larger proteins in sequencing work, which typically involves high efficiency separations using smaller particles with long narrow internal diameter particles to generate the highest possible peak capacity to aid sequence coverage and accuracy of identification, invariably aided by mass spectrometric (MS) detection. OK, for the biochemists out there, I’m one of those bewildered looking small molecule folks, so cut me some slack here.

The biochromatography crew also use ion chromatography with fancy new pH gradients (much quicker than salt gradients apparently) to analyze charged variants. These are proteins and peptides carrying different numbers of fundamental charges that indicates differences as a result of post translational modifications, sequence, or glycan make-up, for example.

They also use size-exclusion chromatography (SEC) (the aqueous version often called gel filtration chromatography [GFC]) to analyze and separate by molecular size and hydrodynamic volume differences that can indicate the formation of protein fragments or aggregates, which have both safety and efficacy implications. Add to this the use of hydrophilic interaction chromatography (HILIC) with fluorescence or (high-resolution) MS detection for the analysis of cleaved and tagged glycan structures as well as hydrophobic interaction chromatography (HIC) for the analysis of proteins in their native (non-denatured state) and for protein purification. That’s not to mention affinity chromatography, which uses antibody-based ligands to capture the protein and then elute to measure the protein “titre” or assay, which is typically used to monitor the production process. So small molecule folks, when was the last time you used so many different techniques in your analysis?
I was being taught new lessons in lecture after lecture and I loved it! For all of you who, perhaps like me, thought that biomolecule analysis was far from “refined”, you can think again. These boys and girls are solving new analytical challenges on a daily basis and they are doing some awesome chromatography. Just what we signed up for many years ago, or perhaps just where we should be headed in out next career move…? Here are some other things I learned during the meeting that will inform my analysis (both small and large molecule) going forward:

Biochromatographers are investigating new methods in ion chromatography using pH gradients (as opposed to salt gradients) to improve the speed, efficiency, and robustness of determination of charged variants.

Reduced column dimensions in ion‑exchange separations are making “high throughput” scenarios more possible.
Using a variety of different detector combinations (static and dynamic light‑scattering with differential viscometry and UV) with SEC can give a huge amount of relative and absolute information regarding molecular size, hydrodynamic volume, and conformation.

The resurgence of hydrophobic interaction chromatography as a very “gentle” but powerful aqueous technique for the analysis of intact proteins, and how this method can be both very rapid and high effective for the characterization of molecules that differ only slightly in their polarity (including “small” molecules).
Highly robust HILIC methods are developed and used in routine analysis of proteins (glycoforms) through a deep understanding of the mechanisms and working principles of the technique. I didn’t once hear during the meeting “We tried that and it didn’t work for us”.

Accurate mass (Fourier transform) mass spectrometers can be used for the analysis of intact protein species to categorize and quantify to very low levels.

Several vendors have released, or soon will release, novel workflows for protein and glycan analysis that save time and make MS analysis of the cleaved, tagged, or digested products possible.

I could go on to discuss all of the “new” things that I learned last week, but I want to keep this column relatively short to help highlight a very important point.

In all of the technical sessions, at the exhibition, and at the social events, I saw chromatographers with that look of excitement that I haven’t seen for such a long time - excitement at learning new things, the opportunity of new possibilities to try out in the laboratory, and the challenge of solving problems. My goodness how I’ve missed this, and how I look forward to increasing my involvement in the area of biochromatography, which I’m now calling The New Frontier in Chromatography.

Reference
1. Incognito, The Column5(2) 12–14 (2009).

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