KOL VOICES | PHARMA RESEARCH

Looking beyond LDL and HDL:

Absolute apolipoprotein profiling for pharma research

For biomarker scientists working across preclinical and clinical development, apolipoproteins offer a more detailed window into lipoprotein biology than routine LDL-C and HDL-C readouts alone. In this Q&A, Tasso Miliotis, Biomarker Lead at AstraZeneca, discusses how targeted mass spectrometry, ApoEdge™ and qRePS™-supported absolute quantification can support reproducible pharmacodynamic biomarker work.

Stakeholder lens	Pharma biomarker development
ProteomEdge technology used	ApoEdge™ – the 18-plex apolipoprotein panel qRePS™-supported absolute quantification
Key publication / use case	Vega et al., “Laroprovstat, the First Oral Small-Molecule PCSK9 Inhibitor for the Treatment of Hypercholesterolemia,” Circulation, 153(25), DOI: 10.1161/CIRCULATIONAHA.125.075973
Application area	Apolipoprotein profiling, cardiometabolic biology, target engagement and pharmacodynamic biomarker research

Q: Can you briefly introduce yourself and your role at AstraZeneca?

A: As a Biomarker Lead at AstraZeneca, I lead the analytical strategy for target engagement (TE) and proof of mechanism (PoM) biomarkers, supporting programs from preclinical research through clinical development. My responsibilities include defining the biomarker context of use, selecting and assessing fit-for-purpose assay platforms, authoring method and qualification reports in accordance with internal and regulatory expectations and ensuring that biomarker plans are scientifically aligned across development stages.

In addition, I provide technical oversight for assay transfer and outsourcing to CROs, including protocol review, troubleshooting and ensuring data quality and assay performance across external laboratories.

Q: From a pharma research perspective, why are plasma proteins and apolipoproteins interesting biological readouts?

A: From a pharma research perspective, plasma proteins and apolipoproteins are interesting because they are accessible, information-rich readouts that can reflect target biology, disease state, and drug effects. Apolipoproteins are particularly interesting because they actively regulate lipid metabolism, inflammation, and related vascular and metabolic processes.

As a result, they can provide useful readouts of disease biology, treatment response, and mechanism of action, particularly in cardiovascular, metabolic, NASH/MASH and kidney disease research.

Q: Many people know LDL and HDL as broad cholesterol categories. What do we miss if we do not look at the wider apolipoprotein system?

A: Looking only at LDL and HDL misses much of the underlying lipoprotein biology. Apolipoproteins provide more direct insight into particle number, lipid transport, receptor interactions, and metabolic regulation. This means they can reveal biological and disease-related changes that are not apparent from cholesterol measurements alone.

“Looking only at LDL and HDL misses much of the underlying lipoprotein biology.”

Q: What is the value of mass spectrometry for quantifying apolipoproteins and related plasma proteins?

A: Mass spectrometry enables selective, multiplexed quantification of apolipoproteins, often with greater molecular specificity than conventional immunoassays. In targeted workflows such as LC-MS/MS with stable isotope-labeled internal standards, it supports robust relative or absolute quantification across a broad dynamic range while distinguishing closely related proteins that may be difficult or impossible to resolve with antibody-based methods.

This is particularly valuable for generating translational pharmacodynamic biomarker data that can be compared across preclinical species and clinical cohorts.

Q: Why is absolute quantification important in a pharma setting where data may need to be compared across studies, sites and time?

A: Absolute quantification is important in a pharma setting because it reports analyte levels in calibrated concentration units rather than assay-specific relative signals, improving comparability across studies, sites, and timepoints. This is essential when integrating datasets across runs, laboratories, or development stages, where relative signals can be affected by batch effects, platform drift, or method changes.

It also enables more rigorous assessment of pharmacodynamic effects, biomarker thresholds, and translation from preclinical to clinical studies. Moreover, absolute values are generally more suitable for assay transfer, standardization and regulatory-facing interpretation than normalized or purely relative readouts.

“Absolute quantification is important in a pharma setting because it reports analyte levels in calibrated concentration units rather than assay-specific relative signals.”

Q: What made ApoEdge relevant, and how did the panel fit into the workflow?

A: The ApoEdge panel is relevant for cardiovascular and metabolic research that is conducted at AstraZeneca Gothenburg. The 18-plex panel is attractive for studying disease mechanisms, pharmacodynamic responses and translational biomarkers, particularly where a more detailed characterization of cardiometabolic biology is needed.

The ApoEdge panel comes in a 96-well plate format where the isotopic labeled protein fragment standards are vacuum dried, allowing for direct addition of sample and reagents and immediate compatibility with automated liquid-handling systems. The standards match the expected dynamic range for plasma apolipoproteins. The setup ensures that standards are present from the start of digestion, minimizing variability introduced by manual or robotic handling, making it ideal for large-scale, high-throughput plasma analysis.

Q: What level of reproducibility do you need, and how does measuring multiple peptides with qRePS affect confidence?

A: A targeted MS workflow becomes useful in pharma research when its technical variability is low enough that true biological or pharmacodynamic effects can be resolved from analytical noise. In practice, this often means CVs <15% for most analytes, with <20% sometimes acceptable in exploratory settings, provided the assay is otherwise fit for purpose in terms of accuracy, linearity, stability, and control of batch and matrix effects.

Measuring multiple peptides per protein increases confidence by confirming identity and quantification across peptides and helping detect peptide-specific artifacts. qRePS further improves confidence by combining peptide-level data into a more robust protein-level estimate, improving precision, reproducibility, and resilience to outlier peptides.

Q: What kinds of decisions, hypotheses or development questions could a robust apolipoprotein panel support?

A: A robust apolipoprotein panel can help test hypotheses on target engagement, pathway modulation, PK/PD relationships, on-target versus off-target effects and compound differentiation. Additionally, it may also help biomarker selection, translational bridging, patient stratification and monitoring of safety-relevant metabolic liabilities across preclinical and clinical studies.

Q: How was ApoEdge used in the Phase 1 laroprovstat study?

A: In the Phase 1 laroprovstat study, ApoEdge™ was used as a mass spectrometry-based apolipoprotein profiling platform to extend pharmacodynamic assessment beyond routine lipid endpoints. Alongside standard measures such as LDL-C, total cholesterol, and non-HDL-C, it enabled broader apolipoprotein profiling, giving a more resolved view of treatment effects on atherogenic lipoprotein biology.

Importantly, ApoEdge detected decreases in ApoB, ApoE, and Apo(a), showing protein-level changes in lipoprotein pathways that are not fully captured by LDL-C and HDL-C alone, and thereby providing a more mechanistic pharmacodynamic profile of PCSK9 inhibition with laroprovstat plus rosuvastatin.

Q: What would you say to a team considering a move from discovery proteomics toward targeted, absolute quantification?

A: Moving from discovery proteomics to targeted, absolute quantification shifts biomarker work from hypothesis generation to decision-enabling measurement. Targeted assays provide the specificity, precision, and calibrated concentrations needed for PK/PD, cross-study comparability and translation in pharma.