Name: Strategies for ADC bioanalysis

Description: Strategies for ADC bioanalysis

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Upload Date: 2025-07-18T00:00:00.0000000

Transcript: Language: EN.
Segment:0 .
Hello, everyone, and thank you for attending today's webinar on strategies for ADCs bioanalysis. I'm Ellen Williams, digital editor of Bioanalysis Zone, and I'll be your host for today's event. Before I introduce you to our speakers, I'd quickly like to cover a few housekeeping items. So firstly, you'll have the opportunity to submit written questions to today's presenters by typing into the Q&A tab at the side of your window.
You may send in your questions at any time during the presentation. We'll collect these and our speakers will address them offline. Please also let us know your thoughts on today's webinar by tweeting us at bioanalysis zone using the hashtag #BZ Webinars so we're delighted to have Chairi Misrielal, Mackenzie Bentley and Kevin Roback presenting for us today.
Chairi is a project manager at ICON, utilizing her experience to lead and support innovative projects within ICON's bioanalytical laboratories. Mackenzie works as a bioanalytical Services Manager focusing on development, validation and bioanalysis of small and large molecule assays. And finally, Kevin is a principal scientist in the method development and validation group at icon, focusing on developing and validating ligand binding assays to assess the pharmacokinetics, pharmacodynamics, and immunogenicity of large molecule therapeutics and biomarkers.
Thank you again for joining us today, everyone. And I'll now hand over to the ICON team to start today's presentation. Hello, everyone. Welcome to this webinar presented by ICON where we will talk about ADCs. So ADC stands for antibody drug conjugates. And ADCs are drugs that combine very potent toxic payloads with a high selective antibody.
So this antibody guides the payload to its target, where it facilitates selective delivery to specific cells. So one example is where the ADC treatment is used is in cancer treatments, where the ADC is deliver powerful chemotherapy drugs to specific cancerous cells, and this results in damage or destroying cancerous cells without harming nearby cells that are healthy. So there are three main components of ADC.
So you have a monoclonal antibody which binds to a specific antigen on the surface of specific cells. You have the drug or payloads and you have the linker which is the binding agent between the payload and the antibody. So the stability of the linker is an important factor in the ADCs efficacy and safety, because on the one hand, it has to be able to be maintained in different conditions to make sure that a cytotoxic agent can be delivered only at the right place.
But also, on the other hand, it has to be able to release the payload once the antibody has bound to or entered to the target cell. So currently, there are 13 FDA approved drugs for the treatment of hematologic cancer in solid tumors. So over the years, many ADC drugs have been tested, which can be observed by the timeline shown on the left, which is ranging from the 1980s till the 2020.
And this includes the key events and discoveries in ADC research and development. Also on the right graph, you can observe a yearly growth of the number of documents that are related to ADC research and development, which indicates the relevance of these drugs. So at icon we are also actively involved in bioanalytical ADCs assay for drugs in development since 2013. And we also see an increase in the number of studies that are related to ADCs in the recent years.
So here you can see a graph that shows the diseases that are explored in ADC related publications. So on the left, you can see that ADCs are developed and researched in several cancer types, but not only in cancer types. The ADCs are developed also. On the right, you can see that other disease implications are also being explored using ADCs as a therapeutic intervention.
So the development of ADCs faces a lot of challenges due to the complexity of developing this type of molecule for therapeutic purposes, and it is a balancing act between the safety specificity for the target and maximal potency. So, to understand the pharmacokinetics of ADCs and its metabolism to support drug development, it is important to develop accurate and robust bioanalytical methods.
So in this webinar, we will highlight different types of bioanalytical methods to support ADC drug development and their challenges. To give you an overview of the current status of ADCs in bioanalytical assays. So we will discuss the following methods. The liquid chromatography mass spectrometry assays, which is presented by my colleague MacKenzie. And we will present the ligand binding assays which is broken down to the pharmacokinetics and immunogenicity where the pKa part is presented by my colleague Kevin and the immunogenicity is presented by myself.
So with this, I would like to give the floor to my colleague Mackenzie, who will talk more about the LCMS parts. Thank you Chairi. As noted in the previous slide, LCMS may be utilized for various PCA applications for ADCs analysis. The most widely used application for ADCs PCA analysis by LCMS is the quantitation of the free payload. Since the payload or warhead of an ADC is typically a small molecule, LCMS is the go to platform.
Matrix samples are extracted using protein precipitation or solid phase extraction followed by analysis. The payload may be monitored as a standalone free payload assay or the payload attached to the linker. The assay also may be multiplexed with a relevant metabolite. A challenge to consider when developing these assays is ensuring the selectivity, specificity, and stability is tested in the presence of excess ADC.
This testing may be performed during initial development as well as the core validation and the long term stability testing. The validation of the free payload assay follows the MDM guidelines very closely. Another application for ADCs pq analysis is the use of hybrid MS to monitor the conjugated payload. These applications utilize an capture step where the capture reagent is either an anti-idiotypic antibody or the target to capture at the antibody site of the ADC.
After elution from the capture, a forced cleavage step is performed to effectively cleave the payload with or without the linker from the antibody. This is followed by an analysis on the LCMS. A benefit to this application is to gather the da information, as well as being able to multiplex with either the payload, linker and metabolite, or payload and metabolites. A challenge to this is the identification of an effective capture reagent that allows for the high level of selectivity necessary for these assays.
Also, they tend to be more expensive than your standard free payload assay that does not require as extensive cleanup for the front end. The next application is the analysis of total antibody using LC MS. Hybrid LCMS capture for an antigen with an anti-idiotypic or target reagent is also utilized for this application. Followed by an enzymatic digest where the antibody is cut into pre-selected signature peptides.
These signature peptides are unique against the matrix proteoglycans and used for quantitation. This capture step may be skipped if you have a robust signature peptide that doesn't that is unique to the species proteoglycans. And it does not have interfering peaks that come from the matrix itself. A benefit to hybrid LCMS is the high selectivity, and a challenge may be that, again, the finding of a effective capture reagent and the expense.
The final application I'll be discussing is PK analysis of the conjugated antibody by hybrid LCMS. Again, the capture step is utilized. But rather than an anti an anti ID or a target capture, anti payload capture is utilized. Following the capture and digest is performed to cut the antibody down into its signature peptides for quantitation.
For all the hybrid LCMS assays I discussed, validation is based on the application components as well as the endpoints, while icam 10 is closely followed. Some tests may be modified based on scientific justification in order to acquire the applicable data needed. So now I'll pass it over to Kevin Roback to discuss ADC PCA analysis by lba. Thank you, MacKenzie, for giving that overview of the LC MS methods used for measuring the pKa of ADCs.
He said, I'm now going to discuss the ligand binding assays used for measuring the pharmacokinetics of ADCs. Now, ligand binding assays are currently the preferred method for measuring the pKa of the antibody component of the ADC, and the three typical methods that you see are for measuring the free payload via LCMS and the conjugated antibody via lba, and the total antibody measured via lba.
The two assays are utilized to determine any changes to the conjugation of the drug after dosing for the conjugated antibody assay, we'll typically use an antibody against the payload or the linker for the capture step. And then the detection will be an anti-idiotypic antibody or a generic anti-human FC antibody. This method should be specific only to antibody that's conjugated with the drug.
For the total antibody method, we'll use an anti-idiotypic antibody or a target capture. And then we'll use either another anti-idiotypic antibody or an anti FC detection antibody for the detection. And this assay is going to be nonspecific. So we'll pick up all of the antibody backbone including anything that is non conjugated. Now these methods are ligand binding sandwich Elisa's.
And they come with all of the challenges typically seen with other Eliza's for measuring monoclonal antibodies, including selectivity or stability, but there are some special considerations because of the conjugation of the antibody. So for the conjugated antibody assay, you need to ensure that the method is specific for your conjugated antibody and that there is no interference from the unconjugated antibody.
So you can test this by performing interference by spiking high concentrations of unconjugated antibody with a drug antibody ratio of 0, and seeing if that impacts your recovery of the antibody drug conjugate for the total antibody pKa assay. You can have issues with the conjugation of the ADC changing the binding epitopes on your antibody. And this could, because of the heterogeneous nature of the ADC or any changes to the ADC during analysis or after dosing.
The change in conjugation could impact your quantitation, so you want to evaluate the impact of reference standards with different drug antibody ratios, and see how that changes your assay performance. You need to make sure that the antibody is reading the same with a drug antibody ratio of 0, or with your conjugated antibody. However, this assessment can be a little bit difficult or impractical to perform because of the difficulty in obtaining reference standard with well-characterized drug antibody ratios and differing drug antibody ratios.
And then there are some special considerations for both the conjugated antibody method and the total antibody method. Like I mentioned previously, these are still sandwich ELISAs for measuring pharmacokinetics and therefore they fall under the umbrella of ich 10, with potentially adding on additional assessments via scientific justification or to assess the effect of conjugation, as I discussed in the previous slide.
Another thing to consider is in vitro stability of your ADC molecule. You need to make sure that during your analysis, the level of conjugation is not being changed. That can especially impact your conjugated antibody method. So you need to make sure that you're handling the material correctly, and that there is no change in the level of conjugation while you're analyzing the molecule.
Another thing to make sure, and this is something to determine early on in your bioanalytical strategy, is to ensure that the methods performed are adequate to characterize the full metabolism of the drug. So I mentioned before that the three assays you typically see are for free payload conjugated antibody and total antibody. However, there can be more assays to assess. Like MacKenzie mentioned you could assess for conjugated payload or potentially active payload versus inactive payload.
And this all depends on the mechanism of action of the drug and any potential biotransformation of the drug. The FDA guidance also allows sometimes to assess fewer assays to characterize the constituent parts of the ADC, depending on the mechanism of action and data from previous studies. For example, they do allow to not measure free payload. If it's been demonstrated that the free payload is circulating in low enough amounts and cannot be quantitated quantitated by a sufficiently sensitive assay.
There's also allowance in certain situations to only measure conjugated antibody. If it can be demonstrated that the ratio of conjugated antibody to total antibody is consistent. However, this really underpins the importance of discussing your bioanalytical strategy with regulatory agencies to ensure that the proper data is being generated. And now we've discussed in detail the bioanalytical methods for the pharmacokinetics of ADCs via LCMS and LDA.
And now I'd like to pass it back to Chairi to discuss the immunogenicity of ADCs. Thank you, Kevin for giving the overview of the pKa part. So I will now move on with the immunogenicity part. So ADAS can be formed against different parts of the ADC such as the antibody itself, the linker payload, or any of the conjugated induced neoepitopes at the attachment site.
And the FDA guidance recommends that immunogenicity assays should be able to measure responses to all the components of the ADC. So measuring ADAS is generally performed using a bridging electrochemiluminescence immunoassay, which is known as MSD. So the use of different assays for ADAS against the antibody and the payload linker complex will provide the complete picture of the treatment related immunogenic response.
So one of the challenges that need to be taken into consideration for the development of the ADA assay is the complexity and structure. So due to the nature of ADCs, measuring ADAS is challenging because Ada's may form against the antibody, the linker or the payload, which makes the assay development more complicated. So a more pragmatic approach, which is often used, would be to develop one ADA assay for Ada's against the complete ADC linker payload complex, which is shown in panel one for both screening and confirmation tears.
And if needed, the positive confirmed samples can then be further evaluated with more specific ADA isotype characterization to investigate whether the ADA binds to the antibody component. The drug payload, or both. So with direct or indirect assays, these can be used to detect antibodies targeting the payload or the linker. It is important to note that it should be determined upfront if such further characterization essays is needed, because it can take a significant amount of time to generate the relevant critical assay reagents.
So neutralizing antibody assays assess whether the formed ADAS block the therapeutic effects of the ADC. So, unlike the traditional monoclonal antibodies, ADCs act via cytotoxic payloads and therefore the neutralizing antibody assays for ADCs must determine not only the prevention of binding to the target receptor, but also the prevention of payload delivery.
So there are two assay formats that can be used to measure Nab activity, which are the cell based assays and the plate based competitive ligand binding assays. The cell based assays are used to evaluate the functional and neutralizing activities of the neutralizing antibodies by measuring the ability of the ADC to induce cytotoxicity in or to the target cells, and the plate based assays assess whether neutralizing antibodies block the binding of ADCs to their target antigen, and that prevents the ADC from performing its intended therapeutic action.
So, one of the challenges in plate based neutralizing antibody assays for ADCs is cross-reactivity. ADCs are complex, containing both the antibody portion, a linker, and a drug payload, and the neutralizing antibodies may recognize either components, which makes it difficult to design assays that specifically measure the neutralization, whereas the cell based assays are more complex to develop and also to use for sample analysis.
So it is therefore recommended to use the essay format strategy with relevant regulatory authorities as early as possible, to allow sufficient time to produce critical reagents or cell lines, and relevant positive controls to develop the assay in the bioanalytical laboratory. So summarizing in this webinar, we have talked about different bioanalytical assays that are required for supporting ADC drug development, and all these assays have their own limitations and challenges.
So this slide gives a simplified overview of the bioanalytical assays for assessing pKa, ADA, and nep of ADCs. And it is important to start early with defining a strategy for bioanalysis, and also to closely collaborate with cro partners to develop and validate essays that meet the needs of the drug development program.
So with this, I would like to close this webinar, and if there are any questions regarding the content of this webinar, please reach out to one of the presenters via email and we will reply to your questions as soon as possible. Thank you for your time. Thank you very much to Chairi, Mackenzie and Kevin for that presentation, and to everyone who followed along in the audience.
As Chairi mentioned, the ICON team will address your questions offline, so please do continue to submit them into that Q&A tab. You'll receive an email with how to watch this webinar on demand very shortly, but in the meantime, don't forget to visit us at bioanalysis zone.com for more webinars. Thank you again for attending today's session and we'll hopefully see you soon.

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