In the last decade, advancements in technology including innovative engineering processes and enhanced data processing tools have led to the successful development of many novel large molecule therapeutics in the drug development field. In fact, from 2005 through 2019, the number of new biologics approved by the FDA more than doubled every four years. With these successes, many drug developers are leveraging new approaches for design and production of biologic modalities in their pipelines with multifunctional drugs taking center stage. These may include antibody-drug conjugates, antibody-oligonucleotide conjugates, peptide conjugates, and other engineered modalities that combine large and small molecule components within a single therapeutic system. As multidomain therapeutics become more common, the bioanalytical community has begun to recognize that our previous strategies for clinical testing may need to be revisited and adjusted to better fit these complex, novel modalities.
How are large and small molecule bioanalytical approaches different?
Bioanalysis, like therapeutic modalities, has traditionally been divided into two distinct categories: small molecule methods and large molecule methods. Small molecules are low molecular weight compounds with simple, nontertiary structures, typically ranging from 100 to 1000 daltons in size. As such, methods to measure small molecules in blood or other matrices have historically been via LC-MS.
Large molecules are more complex in structure with some weighing over a million daltons. Various challenges including charge states, resolution of dense signal sets, slow diffusion, adsorption, and precipitation, create a major barrier for quantitating biologics using LC-MS approaches. Instead, immunoassays such as ELISA and ECLIA methods have become the standard and preferred approach.
How are bioanalytical strategies changing for complex, multidomain biologics?
As new therapeutics continue to combine structural components of large and small molecules, the boundary between traditional bioanalytical methods for each category of drug has blurred. As such, providing bioanalytical support for complex modalities cannot be approached using small versus large molecule thinking but instead requires a combination of ligand-binding assays, LC-MS, hybrid immunocapture methods, immunogenicity assessments, and complementary characterization methods.
An excellent case study for implementing integrated strategies is the bioanalytical package for conjugate drugs such as Antibody-Drug-Conjugates (ADCs) which we have reviewed in a previous blog. ADC testing strategies can vary widely by drug program as there are many different methods being used for the same measurements. Often the decision to use one method over another is driven by experience or comfort with a particular historical platform. As such, instituting a consistent approach to ADC bioanalysis across the industry will require reshaping our thinking about these approaches. The key to integrated testing will be to start with the biological question, define what we are measuring, then select the appropriate platform or combination of platforms regardless of historical precedence.
Are there any new methods being used for testing these types of therapeutics?
One of the emerging strategies for ADCs includes the use of hybrid LC-MS to bridge the gap between large molecule approaches (immunoassays) for the protein component of the drugs and small molecule approaches (LC-MS) for the small molecule component of the drug. In Hybrid LC-MS, an immunoaffinity capture step is performed which uses a ligand or antibody to specifically enrich the protein component (antibody) of the ADC prior to digestion and analysis via LC-MS. The benefit of this approach is that the total antibody, conjugated antibody, and payload can all be measured in a single assay format.
As with many newer methods, the use of hybrid LC-MS presents several challenges. The first is that developing and optimizing such a method requires technical expertise with both LC-MS and immunoassays, two skills which are often siloed into different groups within scientific organizations. The second, and perhaps more immediate concern, is the limited regulatory guidance around hybrid methods. As LC-MS and immunoassays’ guidelines are typically distinct from one another and have differing requirements including the identification of critical reagents, selection of controls, and acceptance criteria for validation or performance.
Despite these challenges, hybrid LC-MS is an example of how adaptive testing strategies can be designed to provide scientifically driven solutions for the changing landscape of bioanalysis for complex biologics.
What are the main considerations for bioanalytical testing of complex modalities?
The future of bioanalysis will not be defined by choosing between LC-MS and immunoassays, but by knowing how to take a tailored approach to generate a coherent picture of drug exposure, disposition, and biological activity by selecting and integrating the appropriate technologies to answer the scientific questions at hand. This will mean having technical and scientific staff that are well-versed in both small and large molecule strategies, and who can leverage a variety of methodologies across platforms. Often, there are overlapping methods that provide the same measurements so understanding the subtle differences and individual strengths of each potential approach will play a critical role in establishing a robust, comprehensive bioanalytical package.
At Celerion we have extensive expertise with both LC-MS and immunoassay services. To ensure we maintain the level of communication and collaboration required to be successful, these services are offered within the same site at each of our laboratories. Our cross-functional teams continue to provide comprehensive bioanalytical services to clients at all phases of drug development from preclinical to late stage-clinical. Contact us today to learn more about our offerings for complex biologics, including hybrid LC-MS capabilities.
References and Further Reading:
FDA Drug Approvals: https://www.cbo.gov/publication/57126
Wang J, Gu H, Liu A, Kozhich A, Rangan V, Myler H, et al. Antibody-drug conjugate bioanalysis using LB-LC-MS/MS hybrid assays: strategies, methodology and correlation to ligand-binding assays. Bioanalysis. 2016 Jul;8(13):1383-401. https://pubmed.ncbi.nlm.nih.gov/27277879/
Khan RMN, Zeng Y, Lanihun AA, Arisa OT, Horner JL, Figg WD. Recent Advances in Bioanalytical Methods for Quantification and Pharmacokinetic Analyses of Antibody-Drug Conjugates. AAPS J. 2025 Sep 4;27(6):138. https://pubmed.ncbi.nlm.nih.gov/40908395/
Werth EG, Roos D, Philip ET. Immunocapture LC-MS methods for pharmacokinetics of large molecule drugs. Bioanalysis. 2024 Apr;16(7):165-177. https://pmc.ncbi.nlm.nih.gov/articles/PMC11845110/