Protein Quantitation: Finding the Right Assay for Your Needs

The detection and quantitation of proteins, whether as therapeutic agents or biochemical markers, plays a vital role in drug development and biopharmaceutical research. Although small molecules have traditionally dominated the pharmaceutical landscape, the use of large molecule drugs—such as monoclonal antibodies—has seen steady growth over the past two decades and is projected to surpass $1,300 billion by 2033 [1]. As with all pharmaceutical products, understanding the stability, bioavailability, and metabolism of large molecules in-vivo is critical. Proteolysis and cellular processes modify and degrade these drugs over time. Additionally, the route of administration (e.g., intravenous, intramuscular) can significantly influence a drug’s half-life and bioactivity. Together, these factors are central to the study of pharmacokinetics (PK).

Equally important is the study of pharmacodynamics (PD), defined as the effect a drug has on the body. Many monoclonal antibodies are designed to modulate biochemical and physiological pathways, such as suppressing an overactive immune response or inhibiting abnormal cell proliferation. Drug efficacy testing often involves tracking downstream biological markers, for example, reduced pro-inflammatory cytokines after drug infusion. However, large molecule drugs can also trigger immunogenicity responses, leading to unwanted, sometimes life-threatening, side effects. To ensure both safety and efficacy, the behavior of host proteins influenced by the drug, collectively referred to as biomarkers, must be carefully monitored throughout in-vivo studies.

While the need to track a large molecule drug and its biological effects is paramount, there are significant bioanalytical challenges. Human blood contains tens of thousands of proteins, with even higher diversity in disease states. For well-studied diseases, biochemical signatures from prior research can guide analysis. However, rare disease variants or under-studied conditions may lack published data, requiring an untargeted biomarker discovery approach. This can be achieved through high-resolution mass spectrometry and proteomics analysis.

Mass spectrometry (MS) has emerged as a leading technology for biomarker detection and large molecule drug quantitation, offering unmatched sensitivity, specificity, and molecular insight. High-Resolution MS (HRMS) instruments, such as the Thermo Scientific Orbitrap Exploris, and triple quadrupole (triple quad) mass spectrometers provide complementary strengths for PK/PD studies in clinical research. The Orbitrap Exploris, with its high-resolution, accurate-mass (HRAM) capabilities, enables comprehensive biomarker identification by precisely measuring molecular masses, elucidating structures, and detecting post-translational modifications. Once potential biomarkers or drug analytes are characterized, the triple quad MS can be used for targeted quantitation via multiple reaction monitoring (MRM)—ideal for tracking large molecule drugs in complex biological matrices.

This discovery-to-quantitation workflow offers high specificity, multiplexing capabilities, and low detection limits, making it well-suited for PK/PD applications. Figure 1 shows a side-by-side comparison of how HRMS and triple quad MS are typically applied in bioanalytical method development. However, challenges such as extended method development times, complex large molecule sample preparation, and the significant investment in instrumentation must be considered, which is why outsourcing to a lab with extensive experience and capabilities is crucial. Importantly, MS-based PK/PD methods must be rigorously developed and validated in accordance with ICH M10 and FDA bioanalytical method validation guidelines to ensure regulatory compliance.

Figure 1: Comparison of typical bioanalytical applications: HRMS versus Triple Quad MS.

Alternatively, once biomarkers have been identified, another widely used approach is ligand binding assays (LBA). Within LBA, the most common method is the Enzyme-Linked Immunosorbent Assay (ELISA). In ELISAs, a capture antibody binds the target—whether a large molecule drug or biomarker—in a flat-bottom plastic microplate. The capture antibody is then bound by a detection antibody linked to an enzyme such as horseradish peroxidase (HRP), which produces a measurable signal (color change or fluorescence) upon substrate addition. The signal intensity correlates with the target concentration via a standard curve. ELISAs are cost-effective, highly sensitive, and automatable. For biomarker analysis, there is a wide range of commercially available antibodies and ready-to-use kits, simplifying assay development. The method is well-established and widely accepted by regulatory agencies like the FDA.

ELISAs are often a starting point for protein quantitation in large molecule drug discovery. However, limitations include matrix effect interference, narrow dynamic range, scalability challenges, multiple wash steps, and long incubation times. The Meso Scale Discovery (MSD) platform overcomes many of these drawbacks and is widely used from early discovery through clinical trials. In MSD assays, testing is performed in an electrode-lined microplate. A capture antibody and target are added as in ELISA, followed by a detection antibody linked to a ruthenium (Ru) label. When near the electrode, electrical stimulation excites the Ru label, generating a light signal detected by the MSD instrument’s camera. Figure 2 illustrates the difference between ELISA and MSD detection.

Figure 2. ELISA signals are generated by the activity of an enzyme tag (e.g., HRP) on a substrate, resulting in a color change in the microplate well. In MSD, the Ru tag is excited by the electricity passing through the electrode and produces a light signal.

Although MSD has a higher upfront cost, it offers advantages such as a wider dynamic range, smaller sample volume requirements, higher specificity, reduced matrix interference, shorter assay times, and the ability to multiplex up to 10 targets in a single well. The platform also offers validated kits and customizable panels for PK/PD studies. As with ELISA, validated MSD methods can be submitted to the FDA as part of large molecule drug development packages.

All of these protein quantitation methods: HRMS, triple quad MS, ELISA, and MSD, are available at Emery Pharma. With our extensive expertise, we can help select the method best suited to your biopharmaceutical research, whether in early-stage discovery or clinical trials. The table below summarizes each method’s performance and cost considerations.

Table 1. Comparison of three protein quantification methods based on performance and costs.

If you are developing a novel large molecule drug or producing a biosimilar, contact us online or call +1 (510) 899-8814 to see how we can accelerate your drug development project!

References

1. https://www.biospace.com/biologics-market-size-to-hit-around-usd-1-37-trillion-by-2033#:~:text=According%20to%20the%20latest%20Research%20by%20Nova,during%20the%20forecast%20period%202024%20to%202033.

About the Author

Authored by Dr. Janet Liu, Director of Biology, and Dr. Prajita Pandey, Associate Director of Chemistry.