Deeper than the definition: ADCs, UHPLC and superficially porous particle technology

While we welcome the exciting 6th edition of The Bioanalysis Glossary, it presents a fine opportunity to jump on the occasion in order to extrapolate a little on a couple of terms that have some meaning beyond what is conveyed, at least personally. So if you will forgive me going to town with this, here are a couple of terms that fall into this bracket.

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1. Antibody–drug conjugate (ADC)

Bioanalysis Glossary definition: Biologically active small molecule (payload) chemically linked to a monoclonal antibody (mAb).

This fascinating modality presents a very contemporary and effective targeted therapeutic delivery format, and associates with a complex multi-faceted bioanalytical challenge. Bristol Myers Squibb (NJ, USA) is proud to be at the forefront of research and development in this domain. How it works on a therapeutic level is a splendid tag-team operation, through harnessing the targeting proclivity of the antibody component together with the potency of the subsequently-delivered payload, typically a number of small molecules attached initially to the antibody.

Bioanalytically, it comes with a mini-maelstrom of requirements, while conveniently bolstering understanding and strengthening communication between ligand binding scientists and LC–MS scientists. While large molecule bioanalysis has traditionally been within the remit of ligand binding groups, triple quadrupole mass spectrometers have become important workhorses for these endeavors, harnessing immunocapture in the preparation where necessary. Plus, there are occasions where high-resolution mass spectrometry comes as a celebratory cherry on top.

To get to the nitty-gritty, we need the measurement of free payload, total antibody, total ADC, and ADA/immunogenicity. In addition to these initial measurements, when we encounter the likes of instability of linker and need to investigate, we then need to bring in the measurement of the drug-to-antibody ratio, the DAR. This is approached in terms of what is referred to as DAR shift, pertaining to the shedding of payload over time in vivo, and it’s also the part that is most conveniently done with high-resolution mass spectrometry, through which we can measure the different m/z species fully resolved and via mass spectral intensity.

Additionally, there is a useful formula for average DAR, utilizing total ADC over total antibody and multiplying this by the initial DAR, but this must be attained with DAR-sensitive methods. This means essentially adhering to careful methodological design as regards to capture and detection, the latter normally being mass spectral. Similarly, the significance of binding reagents should not be underestimated, as the effectiveness of such methods relies heavily on reagent availability and suitability. That being said, could the day be coming, somewhere on the horizon, where such reagents are possibly no longer always required…?

2. Ultra-high-performance liquid chromatography (UHPLC)

Bioanalysis Glossary definition: An LC separation technique using support particles that often have diameters less than 2 µm in size. These smaller particles make it possible to obtain more efficient separations but also increase the pressure that is required to apply the mobile phase through a given length of column.

There have not been many more significant advancements in LC coupled to any kind of detection, including mass spectrometric, than when the so-called UHPLC technology became a commercial reality in the mid-2000s. It was a monumental step forward for resolving power and sensitivity in quantitative methods. Exhaustive run times could be substantially lowered, to boot. The key, indeed the inspiration, lies in the use of very small and uniform particles in the column packing, going to 1.7 micron rather than what was normal previously for analytical, at 3 to 5 micron.

Furthermore, the pertinent technology was perfected that would allow front-end systems to readily cope with the back-pressure increases, up to the order of ten-fold as compared to humble HPLC. It was also ensured that absolutely minimal extra-column volume was present, especially challenging in the injector system. And thus it became, as alluded to, a joyous commercial reality. In the packed columns themselves, smaller particles result in the higher back-pressure, naturally, but this is the beating heart of the improved output. The reduced pore length, not to be confused with pore diameter, in these particles offers far less broadening due to slashing down the well-known mass transfer phenomenon, as per van Deemter, a name shudderingly familiar to all who have studied chromatography.

In fact, we could move up to any practical flow rate and expect no significant deterioration in chromatographic peak width, something that would definitely be detrimental in larger particle columns with accordingly longer pore lengths, as we moved to higher flow rates. Therefore, working well under pressure, a concept easily translatable from other contexts, is fundamental to success!

The same key principle applies in perceiving the benefits of superficially porous particle columns, also known as core-shell technology…

3. Superficially porous (core-shell) particle technology

Bioanalysis Glossary definition: In LC columns, the use of particles that have a nonporous core but fully porous outer layer.

The non-porous core effectively reduces the pore length to that of the shell alone, so we realize that same chromatographic set of benefits, but the disadvantages of ultra-high pressure do not have to weigh in under these circumstances. Hence, if you are not aboard the core-shell locomotive, I think it’s time to become a fan of the superficial. There are no holes in this technology, apart from the obvious! All of which makes me wonder, almost philosophically, about the bigger picture…

There we have my modest collection of definitions with a dollop more depth. Look out for more in-depth deliberations from another set of hands!

Disclaimer: the opinions expressed are solely that of the author and do not express the views or opinions of their employers, Bioanalysis Zone or Taylor & Francis Group.