hilst quantifying mRNA provides an indication of payload delivery, measuring tissue mRNA concentration alone is insufficient to understand therapeutic efficacy. In this interview, the QPS Holdings, LLC (DE, USA) team addresses critical bioanalytical strategies and regulatory considerations for lipid nanoparticle (LNP)-mRNA therapeutics, highlighting the importance of measuring both mRNA delivery and functional protein expression.
Meet the Subject Matter Experts
Zamas Lam
SVP and Global Head of Bioanalytical & Preclinical Development
QPS Holdings, LLC (DE, USA)
Zamas is the Senior Vice President of Preclinical Development at QPS (DE, USA) and is trained in mass spectrometry, carbohydrates and glycoproteins. Zamas is one of the world’s few high-resolution mass spectrometrists by training and by trade, with a passion for biologics mass spectrometry and gene therapy. Since graduate school, he keeps thinking that it will be fun to work on a gene level in drug discovery and development versus at the protein level or at the metabolite level!
Aurora VanAuken
Market Research Intern, GBA
QPS Holdings, LLC (DE, USA)
Aurora is currently a Market Research Intern for GBA at QPS and a fourth-year Biomedical Engineering student at the University of Delaware. She brings 3 years of preclinical research experience focused on the development of polymer and lipid nanoparticles as cancer therapeutics. Her work involved formulation optimization and pharmacokinetics, as well as supporting in vivo studies. At QPS, she contributes to data analyses, pipeline tracking, and identification of business development opportunities across global markets. Outside of science, Aurora enjoys distance running, fundraising and reading.
Xun Wang
Executive Director, Bioanalysis
QPS Holdings, LLC
Xun Wang joined QPS in 2009 and is currently serving as an Executive Director of Bioanalysis in the Department of Translational Medicine. In this position, Xun serves as an SME and team manager for developing and validating bioanalytical immunoassays to support biologics drug development, including biosimilars, ADCs and bispecifics. Prior to joining QPS, Xun held Principal Scientist positions at GlaxoSmithKline (PA, USA). Xun received his B.A. in Chemistry from the University of Utah (UT, USA) and his Ph.D. in Biochemistry and Molecular Biophysics from the University of Pennsylvania (PA, USA).
Meet the Subject Matter Experts
Zamas Lam
SVP and Global Head of Bioanalytical & Preclinical Development
QPS Holdings, LLC (DE, USA)
Zamas is the Senior Vice President of Preclinical Development at QPS (DE, USA) and is trained in mass spectrometry, carbohydrates and glycoproteins. Zamas is one of the world’s few high-resolution mass spectrometrists by training and by trade, with a passion for biologics mass spectrometry and gene therapy. Since graduate school, he keeps thinking that it will be fun to work on a gene level in drug discovery and development versus at the protein level or at the metabolite level!
Aurora VanAuken
Market Research Intern, GBA
QPS Holdings, LLC (DE, USA)
Aurora is currently a Market Research Intern for GBA at QPS and a fourth-year Biomedical Engineering student at the University of Delaware. She brings 3 years of preclinical research experience focused on the development of polymer and lipid nanoparticles as cancer therapeutics. Her work involved formulation optimization and pharmacokinetics, as well as supporting in vivo studies. At QPS, she contributes to data analyses, pipeline tracking, and identification of business development opportunities across global markets. Outside of science, Aurora enjoys distance running, fundraising and reading.
Xun Wang
Executive Director, Bioanalysis
QPS Holdings, LLC
Xun Wang joined QPS in 2009 and is currently serving as an Executive Director of Bioanalysis in the Department of Translational Medicine. In this position, Xun serves as an SME and team manager for developing and validating bioanalytical immunoassays to support biologics drug development, including biosimilars, ADCs and bispecifics. Prior to joining QPS, Xun held Principal Scientist positions at GlaxoSmithKline (PA, USA). Xun received his B.A. in Chemistry from the University of Utah (UT, USA) and his Ph.D. in Biochemistry and Molecular Biophysics from the University of Pennsylvania (PA, USA).
Rationale for dual assessment
Why should LNP-mRNA biodistribution studies include both mRNA exposure and encoded protein expression?
For LNP-mRNA therapeutics, tissue mRNA concentration is an important measure of payload distribution, but it is not a direct surrogate for pharmacologically relevant expression. The relationship between mRNA exposure and encoded protein production may be influenced by LNP uptake, endosomal escape, intracellular release, mRNA stability, translational efficiency, and local protein turnover. Consequently, mRNA and protein profiles may differ substantially in magnitude, tissue distribution and temporal persistence.
A scientifically robust biodistribution assessment should therefore distinguish delivery of the nucleic acid payload from productive translation of the encoded protein. Concurrent evaluation of mRNA and expressed protein supports interpretation of exposure-response relationships, target-tissue engagement, pharmacologic persistence, and potential off-target expression liabilities.
Are circulating protein concentrations sufficient, or should tissue-level encoded protein expression also be assessed?
Serum or plasma protein measurements are useful for evaluating systemic exposure, apparent pharmacokinetics (PK), and circulating pharmacodynamic (PD) mediators; however, they generally do not provide adequate resolution of local expression within target and non-target tissues. For non-secreted or locally acting proteins, circulating concentrations may be minimally informative or biologically disconnected from tissue pharmacology.
Tissue-level expression should be considered when local PD activity, organ-specific toxicity, or off-target translation could influence the nonclinical risk assessment. Tissue expression may vary because of organ-specific LNP tropism, cell-type uptake, endosomal escape, translational capacity, secretion, proteolysis, and clearance mechanisms.
A defensible biodistribution strategy typically integrates measurements of LNP-associated components, mRNA and encoded protein across relevant biological matrices. These matrices could include plasma or serum and selected tissues such as liver, spleen, injection site, draining lymph nodes, gonads and any pharmacologically or toxicologically relevant organs. Tissue protein data provide direct evidence of productive expression and help contextualize mRNA biodistribution findings.
From a regulatory perspective, tissue expression data can strengthen the rationale for species selection, dose selection, sampling intervals, PK/PD modeling and off-target risk characterization. Such data are most informative when generated using qualified or validated fit-for-purpose methods with appropriate matrix qualification and documented assay performance.
Study design and bioanalytical strategies
What scientific and regulatory factors should guide the quantification of encoded proteins in biodistribution studies?
Encoded protein quantification should be designed to answer a defined development question. This could be whether the LNP-mRNA product produces the intended protein in target tissue, if expression occurs in safety-relevant off-target tissues, or whether protein expression aligns with PD or toxicologic findings.
Key design considerations include:
- Model selection: species, sex, group size
- Dosing parameters: dose level, route of administration
- Sampling strategy: sampling schedule, tissue panel, matrix availability
- Biological factors: endogenous background, analyte stability, expression kinetics
Species selection is particularly important because LNP tropism, innate immune activation, tissue architecture and translational efficiency may differ between nonclinical species and humans.
Assay strategy should be aligned with the expected concentration range and the intended regulatory use of the data. For Good Laboratory Practice toxicology support or regulatory decision-making, method validation or qualification should address specificity, sensitivity, precision, accuracy where applicable, dilutional linearity, parallelism for ligand-binding assays (LBAs), selectivity, stability, and tissue-specific matrix effects.
What are the principal bioanalytical challenges for encoded protein detection, and how can LC–MS/MS and LBAs be applied?
Encoded protein concentrations may be low, transient and heterogeneously distributed, particularly in off-target tissues. Endogenous homolog interference — whereby closely related native proteins cross-react with assay reagents — and non-specific binding can produce false signals and lead to skewed quantification. Matrix-dependent recovery, proteolytic degradation, limited tissue mass, sample homogenization variability, and contamination during tissue collection or processing are also key challenges.
Tissue matrices are analytically complex and may contain high-abundance proteins, lipids, nucleases, proteases and other components that affect extraction efficiency and assay signal. Matrix-specific assessment is therefore critical; performance demonstrated in plasma or serum should not be assumed to translate to tissue homogenates.
LC–MS/MS can provide peptide-level specificity, multiplexing capability and confirmation of analyte identity when suitable signature peptides, digestion conditions and enrichment strategies are established. LBAs can provide sensitive, higher-throughput quantification when critical reagents are specific and well characterized. Immunoaffinity capture enrichment, hybrid IA-LC–MS/MS workflows, and orthogonal confirmation can improve selectivity and sensitivity in complex tissue matrices.
Functional interpretation and regulatory compliance
Why might mRNA and encoded protein abundance be insufficient to establish functional outcome?
mRNA exposure and encoded protein abundance are necessary but insufficient to establish pharmacologic function. Biological activity also depends on correct protein folding, processing, post-translational modification (PTM), subcellular localization, secretion, target engagement and interaction with downstream pathways. PTMs such as glycosylation, phosphorylation, acetylation, disulfide bond formation, proteolytic processing or lipidation may materially affect receptor binding, enzymatic activity, serum half-life, immunogenicity, tissue localization and overall efficacy.
An encoded protein may be detectable yet functionally inactive if it is misfolded, truncated, improperly modified, mislocalized or rapidly neutralized. Conversely, low-level expression may be pharmacologically meaningful if the protein is highly potent, appropriately modified, localized to the relevant compartment, or capable of amplifying downstream signaling.
Functional interpretation should therefore integrate LNP distribution, mRNA exposure, encoded protein expression, bioactivity or target-engagement assays, PD biomarkers and toxicology findings. This weight-of-evidence approach supports more reliable conclusions regarding efficacy, safety margins, dose selection and clinical translation.
What reference materials and regulatory guidance should support validation of PK and tissue expression assays for encoded proteins?
The preferred reference material is a well-characterized recombinant protein that is representative of the mRNA-encoded product, including relevant sequence, folding, PTMs and biological activity where applicable. Mammalian expression systems (e.g., CHO or HEK293 cells) are appropriate when glycosylation, disulfide bonding, proteolytic processing, conformational epitopes, or other efficacy-relevant PTMs are important to assay performance or biological interpretation.
Reference standards and critical reagents should be characterized for identity, purity, concentration assignment, potency or bioactivity when relevant, stability, lot-to-lot consistency, suitability in the intended matrix, and comparability of efficacy-relevant PTM profiles. Documentation should support traceability and justify use of the material as a calibrator, quality control material or assay control.
ICH M10 provides the primary harmonized framework for bioanalytical method validation and study sample analysis, including expectations for chromatographic- and LBA-based methods used to support regulatory submissions. Relevant validation elements include accuracy, precision, selectivity, sensitivity, calibration model, carryover, dilution integrity, stability, incurred sample reanalysis where applicable, and matrix effects.
For non-clinical biodistribution, ICH S12 provides harmonized recommendations for study design and interpretation of gene therapy biodistribution data, including considerations for test article, species or model, dose level, route of administration, sampling time points, tissue collection and assay methodology. The US Food and Drug Administration and the European Medicines Agency gene therapy guidances should also be considered when defining the regulatory context for biodistribution, persistence, expression and pharmacologic activity.
Together, appropriately characterized reference materials, validated or qualified fit-for-purpose assays, and alignment with current regulatory guidance support credible PK/PD interpretation, nonclinical safety assessment, and regulatory submissions for LNP-mRNA therapeutics.
