Large Molecule Bioanalysis using LC/MS

Introduction to Large Molecules

Therapeutics are an ever-evolving landscape of modalities which range in complexity but can be grouped into two broad categories: small molecules and large molecules. Small molecule therapeutics have historically dominated the marketplace and currently account for roughly 75% of all pharmaceutical drugs. The first therapeutic classified as a large molecule biologic to be approved by the FDA was recombinant human insulin in 1982. The first FDA approval of a large molecule antibody therapeutic, the murine antibody Muromonab-CD3 (OKT3), quickly followed in 1986. Since then, large molecules have become increasingly prevalent in drug development pipelines with more recent successes including many products with worldwide recognition such as the top-selling drug Humira which is an antibody-based therapeutic for treatment of rheumatoid arthritis. Examples of large molecule therapeutics are:

  • Monoclonal antibodies
  • Bispecific antibodies
  • Antibody-drug conjugates (ADCs)
  • Recombinant enzymes
  • Polypeptides
  • Adoptive cell therapies (ex. Chimeric Antigen Receptor or CAR T cells)
  • Viral vector gene therapies (ex. Adeno-associated Virus or AAV)
  • Non-viral vector gene therapies (ex. Lipid Nanopartical or LNP)
  • Recombinant cytokines and growth factors

Large Molecule Bioanalysis

Preclinical and clinical testing of any therapeutic requires specific strategies to evaluate the pharmacokinetics (PK), safety, and efficacy of the therapeutic after administration. However, the bioanalytical methods and platforms used to support large molecules have historically been distinct from those that are commonly applied to small molecules. For example, PK assessments of large molecules commonly leverage ligand binding assay (LBA) platforms such as ELISA (enzyme-linked immunosorbent assay), MSD-ECL (electrochemiluminescence), Gyros, and Luminex. In contrast, small molecule PK assays typically use Liquid Chromatography-Mass Spectrometry (LC/MS).  

LC/MS is a technique that involves the physical separation of target compounds (or analytes) by liquid chromatography followed by their ionization and detection by mass spectrometry. LC/MS can be used for separation, identification, and quantification of both unknown and known compounds as well as elucidation of the structure and chemical properties of different molecules. Recent advancements in LC/MS technology with improved bioinformatics have enabled the application of this technique to new bioanalytical areas to support large and complex modalities. In particular, there has been an increase in the use of LC/MS as an alternative to LBA for large molecule PK assessments, supporting such modalities as monoclonal antibodies, polypeptides, and ADCs.

LC/MS for Large Molecule PK

While both LBA and LC/MS methods are considered appropriate platforms for large molecule quantitation, there are several key aspects of LC/MS that make its selection over LBA advantageous in specific situations. LC/MS has the ability to monitor multiple signature peptides throughout the protein structure which allows multiplexing during analysis for differentiation between the drug and its metabolites or between protein isoforms, distinctions which can’t always be made with a traditional LBA or immunoassay. LC/MS is highly selective which aids in the assay performance when a therapeutic is homologous to endogenous protein(s). It also has a large dynamic range (4 orders) and can usually be implemented much faster than traditional LBA methods.

When developing an LC/MS assay for large molecule analysis, the simplest workflow is a ‘direct digestion’ approach that involves digesting the sample using a protease, such as trypsin, followed by LC/MS-based detection of a single signature peptide. However, to improve the sensitivity of the assay for low-concentration proteins, addition of an immunoprecipitation step can be performed prior to proteolytic digestion to develop hybrid immunoaffinity LC/MS assays. In the hybrid immunoaffinity LC/MS assay format, the drug is captured from the matrix/patient sample by a capture antibody or ligand attached to a magnetic bead or other solid support. Generic capture reagents can be used for the enrichment step which provides a solution for drug programs that cannot source specific antibodies or targets, reagents which would be required for the LBA approach. The addition of automation systems from a pipetting and immunoaffinity extraction standpoint, as well as better data processing platforms, have made high-throughput applications achievable for using LC/MS methodologies to analyze the large sample numbers typically associated with late-stage clinical trials.

Large Molecule Bioanalysis at BioAgilytix

BioAgilytix offers discovery, non-regulated, and GxP-compliant LC/MS bioanalysis to support all stages of drug development from early preclinical through late-phase clinical studies. Our LC/MS and LBA-based scientists are experts in the bioanalytical space and are co-located, allowing our teams to take a collaborative approach to designing the optimal LC/MS and LBA solutions for our clients. We provide de novo method development or can adopt research-grade assays from discovery. We provide method optimization and validation as well as sample analysis and report generation. Advancing your large molecule requires access to specific scientific expertise, alignment with changing regulatory requirements, and advanced platforms.  Speak to one of our scientists today to learn how our expanded lab capacity, advanced LC/MS instrumentation, automation capabilities, and deep expertise in the LC/MS space can be leveraged to support your large molecule program.