Guiding Decisions throughout Drug Development

This talk was presented as a part of the Bioprocess Quality, Data and Analytical Solutions webinar with BIO-EXPO. Hear from Maggie Ostrowski, PhD, Sr. Manager, CE-MS Product Management, about how SCIEX can help guide your decisions throughout drug development, including reducing analytical bottlenecks, risks, and late-stage failures; increasing efficiency; and increasing molecular knowledge earlier in the pipeline.

Maggie takes the audience through:

SCIEX Instrumentation, including recent launches, aligned with the drug development process
The Intabio ZT for characterization, clone selection, cell line development, and bioprocessing
The core technology of the Intabio ZT
Data generated by Intabio ZT including 3 different monoclonal antibody examples
Biologics Explorer Software
Customer case studies

Transcript

The center is from SCIEX. She is Maggie Ostrovsky, senior manager CEMS product management. She's going to share how SCIEX can help guide your decisions throughout drug development, including reducing analytical bottlenecks, risks, and late stage failures, increasing efficiency and increasing molecular knowledge earlier in the pipeline. Welcome, Maggie. Hi.

Great. Thank you, Perry. Hi, everybody. Thanks so much for that introduction. So I'm going to talk today about technologies to reduce analytical bottlenecks throughout drug development. So SCIEX is known for CE and mass spec technologies. We recently launched a multi-capillary CE system, the BioPhase 8800. That covers CESDS, ICIF, glycan identification, DNA RNA purity, and protein purity. On the right-hand side is the Zenotof 7600 mass spec for in-depth characterization of biologics, including for peptide mapping, glycan analysis, and post-translational modification identification with some really exciting novel fragmentation capabilities that allows you to see post-translational modifications that are sometimes missed with other technologies.

But what I really want to talk about today is the Intabio ZT system in the middle there. This is our most recent innovation, and actually it couples CE and mass spec technologies, and specifically image capillary isoelectric focusing, both with UV detection for quantitation, as well as mass spec for identification of charge variants, which has been a challenge in the industry for many, many years. And so really our goal is to provide in-depth information much earlier in the process, and specifically on intact protein therapeutics. Because at the intact level, you're able to assess the heterogeneity of the molecule, and it's a much more clear, straightforward way to understand what's happening with the fermentation process and make the right decisions along the way, with the goal of shortening timelines for development.

And especially as molecules are getting more and more complex with new modalities, the analytical tools really do require new ways to assess the complexity of these molecules. So this is the Intabio ZT system shown here, and that system, what I'm going to talk about is really what we see as a way to fully characterize intact proteins 95% faster than traditional fraction collection approaches with LC-MS analysis. So the conventional workflow requires either CIEF or more typically ion exchange chromatography, method development, fractionation, and then taking those fractions and doing LC-MS analysis.

And then of course relaying those mass spec data back to the original fractions is often a challenge. So we do this directly with the Intabio ZT system, and the goal there is to identify the post-translational modifications to help assess the safety and efficacy of the biotherapeutic throughout the various stages of the development process. And I'll show at the end various examples of how our customers and collaborators have used the Intabio ZT system throughout clone selection, cell line development, and bioprocessing.

I think we all know that protein biologics are heterogeneous because of the post-translational modifications that are imparted during the growth and development process, but also need to be assessed during stability studies, formulation development, and so on. So the Intabio ZT system, really the kind of core technology behind it is an integrated chip-based microfluidic technology that couples the capillary electrophoresis separation within that chip channel, as well as the electrospray tip that ionizes the proteoforms into the mass spec for mass detection. So fundamentally, image capillary isoelectric focusing separates intact proteins or proteoforms based on their isoelectric point, which is a very high-resolution approach to separating intact proteins.

So the entire separation channel is filled with the sample. The electric field is applied within the Intabio ZT system. The proteoforms are separated within an amphilate gradient, so that's all imaged in real time, and that's very useful for assessing the optimal conditions there. Following the focusing, the sample, the electric field is switched and the entire separation is then mobilized to the electrospray tip, and the masses are detected within the Zenotof 7600 system. So this shows you the type of data that the system is able to collect. On the left is the UV electrophoregram with the proteoforms separated within that PI range of 7 to 10. The blue is the mobilization electrophoregram, so that's what we detected at the very end, right before the samples go into the mass spec. And then in green, you see the mass spec data that's generated. This is the mass spec base peak electrophoregram.

You can see that the profiles are highly consistent. The mass spec data is inverted from the UV data because as the mobilization occurs, those high PI proteoforms are detected first within the mass spec. So if we go to the next slide here, what I'm showing are three examples, three different monoclonal antibodies at various PI ranges there, and you can see the consistency between the UV results and the mass spec results.

So each sample, it's about 15 minutes to collect all of this data so significantly faster than other methods and highly integrated, and a platform method. So in general, most of the UV and mass spec conditions are the same across various molecules, which makes it very easy to set up. So if we look specifically at one example, you can see the UV data on the left and the acidic main and basic variants are separated there.

And then in the mass spec data, we look at the mass data for each scan that's collected. And we can see the identification and differences across those proteoforms. So we can see various modifications and heterogeneity of the molecule, including deamidation, sialylated glycans, succinamide intermediate to deamidation, as well as others such as glycation and lysine variants. Now all of the data are analyzed with Biologics Explorer software. And so the Biologics Explorer software provides a three-dimensional view of the entire sample heterogeneity shown in the heat map there in the middle.

And so the analysis is done based on looking at main peaks and the mass differences across the various proteoforms within the sample. So in general, the system increases molecular knowledge earlier in the development process. The intact protein analysis with the Intabio ZT system can be used to identify what's under each peak in the CIEF profile can be used for lead candidate screening early in the cell line, clone selection and cell line development process. It can be used for antibody drug conjugate work to really understand what's contributing to the charge heterogeneity, as well as quick identification of unknown charge variant peaks during process development or even a peak investigation that sometimes occurs in manufacturing. We did recently launch a subunit analysis with the Intabio ZT system. So that helps with identifying sources of charge heterogeneity and helping to localize some of that charge variability very quickly to help assess whether those modifications will impact other processes, to help increase product knowledge with that localization information, and help get information to make decisions on the analytical next steps and help really reduce the risk mitigation strategies during development in CMC. So I want to give a few examples, some of the collaborators and customers we've worked with.

You can link to these particular webinars. Kristen Schultz-Kuzak from AstraZeneca used the Intabio ZT system to assess the antibody drug conjugate, help understand what the charge variant peaks were resulting from. Jonson is using the Intabio ZT system in their clone selection process to help make better decisions on what candidates to move forward with. Maurice says, J &J is using the Biophase 8800 system for protein purity and Greg Adams at Fuji Film is using the Intabio ZT system to assess the process development and so you can have a look at all of these presentations. So with that, I will wrap it up and I'm happy to take questions.

Thanks Maggie, really great stuff. So we have two questions about the Intabio system, Intabio ZT. So the first, we'll take them one at a time. The first is what are the training requirements?

So the Intabio ZT system is straightforward to set up. It's a platform method so all of the reagents are provided and so it's a pretty plug and play method on both the CE and the mass spec system.

The second is what PTMs can be identified at the intact level on that system?

So that's a great question. Actually, there's a number of post-translational modifications because the CIF separation separates the proteoforms based on charge and so that combined with the mass spec information we've seen working with collaborators a number of modifications. Not only can you see the modifications, the charge-based modifications, but also neutral modifications such as various glycosylation structures and so forth. We've also seen things like sequence variants, for example.

We've seen clips. We've also seen structural information such as reduced thials in addition to the kind of more commonly thought of modifications. And looking at that at the intact protein level simplifies things quite a lot compared to looking at things at the peptide mapping level where you're introducing additional complexity from the digestion of all of the combined proteoforms.

You mentioned that you had done some subunit workflow analysis. So the next question is about that. Can you speak to the benefits of the Intabio subunit workflow?

Yeah, so when we do the subunit analysis, it's based on the reduction and we can also use enzymatic digestion using things like fabricator. So the subunit workflow allows for the subunits to be separated and then mobilized into the mass spec. So that helps assess the localization of the post-translational modifications.

So for example, whether it's in the FC or whether it's in the fab. Interestingly, we can also assess structural information because the subunits remain intact in the CIF separation because it's a non-denaturing environment. And then when we do the mobilization, that's a denaturing environment. So we can see, for example, how things are associated and dissociated in the mass spec. So that helps assess things like stability.

I think we have about 30 seconds left. One more question. What role does aggregation play in the sample?

That's a great question. Yeah, so typically in image capillary or isoelectric focusing, there are reagents that can be used if aggregation is a concern, which it is in some cases. So for my mind, for example, which is a mass spec compatible stabilizer can be used if aggregation is a concern in CIF.

Excellent. Maggie, thank you very much for being with us today. That is Maggie Ostrowski from SCIEX. They're part of Dana Hur. Just thanks so much for your time.