The use of mass spectrometry to aid ADC development

Our speaker, Chris Nortcliffe, Mass Spectrometry Manager discusses how Sterling uses mass spectrometry in junction with our traditional techniques to analyse ADCs and other biologics to get the best quality data on these molecules.

00:00 - Introduction to speaker series
00:41 - Biologics, next gens, ADCs
01:40 - Making ADCs at Sterling
03:19 - Analytical techniques and challenges
04:55 - What is Mass Spectrometry and how can it help?
06:50 - Case study: Is it viable?
09:02 - Mass Spectrometry of various payloads
10:08 - MS compared to HIC
11:20 - Reverse phase PLRP separation
12:06 - Native size exclusion chromatography (SEC) MS
13:16 - Native MS compared to HIC
14:17 - Summary

Emily Hardcastle, Marketing Manager: Welcome to our speaker series, ‘the use of mass spectrometry to aid ADC development’.

Our speaker is Chris Nortcliffe, Mass Spectrometry Manager based at our specialist bioconjugation facility in Deeside, Wales, UK. With that, I'll hand it over to you, Chris.

Chris Nortcliffe, Mass Spectrometry Manager: Thanks for that introduction, Emily. Today, I'd like to talk to everyone about the use of mass spectrometry (MS), to aid in the development of antibody drug conjugates (ADCs). And how here at Sterling, we use mass spectrometry in junction with other traditional techniques to analyse ADCs and other biologics to get the real best quality data on these kind of molecules.

First off, I'd like to talk about the biologics field in general, and where that's headed in the market right now. And we're seeing a real growth in the field going from traditional mAb IgG formats into new architectures.

Now some of these are headed towards smaller features such as just variable domains, things like VNARs, Fab fragments, even down to peptides and cyclic peptides, just taking the very small variable binding regions.

On the other side, we're going towards larger more heterogeneous structures, such as bispecifics, tetra bodies, other such formats that have different protein chains that have different challenges for the analytics of how we analyse these molecules. And on top of all these molecules, we've got the addition of drug conjugates of various kinds coming through that add an extra layer of complexity these molecules. So, analysing these can present different challenges going forward.

Where we fit in with this at Sterling, at our site at Deeside, is that we’re a CDMO and we specialise in the development and analytics of these protein drug conjugates. We have a lot of expertise in a variety of linkers and toxins, so looking at a variety of protein scaffolds, including mAbs and other IgGs; and the smaller things I talked about, like mini bodies, and nano bodies, down to peptide, things like that, with a variety of linkers, including cysteine and lysine links to a lot of toxins that are seen coming through the clinic such as exetcans , PBDs and more traditional things like auristatins. So we have a wealth of expertise, analysing all these kinds of molecules, and conjugating them going forward.

The particular challenge of ADCs I’d like to talk about today, is going through the partial reduction approach making ADCs. So with partial reduction approach, we take our IgG of interest and we do a reduction to break apart the disulfide bonds into their free cysteines, and then we conjugate our payload molecule to these.

This approach is quite well established in the field as it gives a very reliable, robust drug antibody ratio in even numbers such as zero, two, four, six, and eight. Because of the breaking apart of the disulfide chains, when we conjugate these drugs on, we break apart the covalent interacts of the molecule, and the molecules are held together by non-covalent interactions, now there can be a couple of challenges in working out exactly what your DAR ratio is for these molecules.

One technique for the analysis of ADCs is hydrophobic interaction chromatography, or HIC. Now, HIC is a non denaturing technique that relies upon the interaction of our ADC for the stationary phase based upon the hydrophobicity. On the left hand side, we can see a HIC trace with separation of the zero, two, four, six and eight drug conjugations, and we can take the peak area of these peaks to get a DAR ratio.

Now, HIC relies on the absorbance of a molecule via UV so we're seeing and inferring what each peak is rather than being a direct measurement. Relying on inferring of peaks, some molecules might not separate very clearly, or where there are overlapping peaks it can be difficult for some molecules to get a DAR from this technique.

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