The therapeutic potential of mRNA is inseparable from its interaction with the innate immune system. Uncontrolled activation of Toll-like receptors (TLRs), RIG-I, or other pattern-recognition receptors can trigger excessive cytokine release, acute inflammation, or impaired translation – derailing both safety and efficacy.
Early profiling of these responses is essential to select the lead candidate with the most favorable immune profile, support regulatory filings, and streamline CMC development. Our mRNA Immunogenicity Profiling service evaluates the innate immune response triggered by your mRNA construct or delivery formulation using human primary immune cells. We quantify cytokine and chemokine release, measure surface activation markers, and assess innate sensor engagement in a controlled, reproducible assay system. The result is a comprehensive risk profile that informs candidate selection, formulation optimization, and regulatory documentation.
Whether you are a small biotech seeking IND-enabling data or a large pharma refining a pipeline candidate, our service delivers actionable immunogenicity data with project-specific scope and clear reporting.
Key Features
Human PBMC & Dendritic Cell Assays
We expose peripheral blood mononuclear cells (PBMCs) or monocyte-derived dendritic cells (moDCs) to your formulated mRNA under controlled culture conditions. These primary human immune cells provide a physiologically relevant platform to detect innate responses that may translate to clinical outcomes. Cell donor pools can be selected to represent diverse genetic backgrounds or specific HLA haplotypes as scoped.
Multiplex Cytokine & Chemokine Profiling
Using validated Luminex or multiplex ELISA platforms, we simultaneously quantify up to 30+ cytokines, chemokines, and growth factors from culture supernatants. Targets include pro-inflammatory markers (IL-6, TNF-α, IL-1β, IFN-α, IFN-β), chemokines (IP-10, MCP-1, RANTES), and regulatory cytokines (IL-10, TGF-β1). Data are reported as absolute concentrations with fold-change over vehicle controls.
Innate Sensor Activation Assessment
To pinpoint the upstream mechanism, we offer TLR and RIG-I reporter assays for human TLR3, TLR7, TLR8, and RIG-I. mRNA samples are incubated with reporter cells expressing specific sensors, and activation is measured via secreted alkaline phosphatase (SEAP) or luciferase readouts. This module helps differentiate pure TLR7/8 engagement from broader RIG-I activation.
Flow Cytometry for Activation Markers
Surface expression of co-stimulatory molecules (CD80, CD86, CD40) and maturation markers (CD83, HLA-DR) on dendritic cells is measured by flow cytometry. Upregulation of these markers indicates the degree of dendritic cell activation, which correlates with downstream adaptive immune priming. Cell viability is co-assessed to rule out cytotoxicity-driven signals.
Assay Options & Scope
Our immunogenicity profiling is customized to your mRNA construct, delivery system, and study objectives. The following modalities can be selected individually or combined in a single project.
In Vitro Human PBMC Assay: Fresh or cryopreserved PBMCs from healthy donors are transfected or incubated with formulated mRNA. Supernatants and cells are collected at defined time points (e.g., 4–24 h) for cytokine and activation marker analysis.
Monocyte-Derived Dendritic Cell Assay: CD14+ monocytes are differentiated to immature moDCs, then exposed to mRNA. This system provides a pure dendritic cell population sensitive to innate sensor activation and is ideal for mechanistic profiling.
TLR/RIG-I Reporter Cell Lines: HEK293 cells stably expressing human TLR3, TLR7, TLR8, or RIG-I with NF-κB/IRF-inducible reporter are used to deconvolve sensor activation and quantify agonist potency relative to controls.
In Vivo Immunogenicity (Mouse Model – Optional): For projects requiring in vivo context, we offer intramuscular or intravenous administration in mice, followed by serum cytokine/chemokine analysis and splenic DC activation phenotyping at early time points.
Kinetic & Time-Course Profiling: Supernatants can be sampled at multiple time points (e.g., 1, 4, 8, 24 h) to capture peak response kinetics. This is valuable for comparing fast vs. sustained activation profiles and for selecting optimal readout windows.
Workflow
Our immunogenicity profiling follows a structured workflow tailored to your construct and study goals.
Study Design & Sample Receipt: Our scientific team reviews your mRNA construct, formulation, and study goals to define the assay panel, cell type, donor number, time points, and controls. You provide mRNA samples (purified and characterized) plus any delivery vehicle components. A detailed project plan and timeline are issued.
Pre-Assay QC of Test Article: Before cellular assays, your mRNA is tested for endotoxin levels (LAL or rFC) and purity (UV absorbance, gel electrophoresis). Acceptable endotoxin thresholds are set per project to avoid confounding immune activation from contamination.
Cellular Stimulation: Human PBMCs or moDCs are isolated, cultured, and exposed to your mRNA (naked or formulated) at concentrations scoped during study design. Vehicle-only, positive (known TLR agonist), and negative (unstimulated) controls are run side-by-side. Each condition is performed in triplicate.
Multiplex & Flow Cytometry Readout: Culture supernatants are collected and analyzed for cytokine/chemokine levels using a multiplex platform. Cells are stained for activation markers and viability, then acquired on a flow cytometer. Data are compiled and pre-processed for statistical analysis.
Statistical Analysis & Reporting: All raw data are normalized, fold-change over vehicle calculated, and statistical significance assessed (e.g., t-test or ANOVA with multiple testing correction). A comprehensive report includes methods, raw data tables, dose-response curves, heatmaps, and a written interpretation of the immunogenicity risk profile.
Quality Assurance
Our immunogenicity profiling service is designed to support regulatory submissions for mRNA therapeutics. All assays are performed under documented standard operating procedures (SOPs) with full reagent traceability and instrument calibration records.
Key quality elements include: endotoxin and purity QC of each test article, inclusion of reference controls in every run, raw data archives, multi-level review of analysis, and final reports that can be included in IND/IMPD sections. We follow industry guidance on immunogenicity assessment and can adapt to your specific regulatory requirements.
Why Choose Us
Human Primary Cell Expertise: Our laboratory specializes in the isolation, culture, and assay of human PBMCs and dendritic cells from multiple donors, ensuring physiological relevance and donor variability assessment.
Comprehensive Multiplex Panels: We offer validated panels covering key innate cytokines and chemokines (e.g., IL-6, TNF-α, IFN-α, IP-10) with the option to expand to 30+ plex for discovery projects.
Sensor Deconvolution: TLR and RIG-I reporter assays allow you to pinpoint which innate sensor is activated by your construct, supporting rational formulation design.
Regulatory-Ready Reporting: Our reports are structured with methods, raw data, statistical analysis, and risk interpretation, suitable for inclusion in regulatory documentation packages.
Backed by Creative Biogene: All projects are delivered under Creative Biogene’s ISO-aligned quality system, with dedicated project management, milestone-based reporting, and full intellectual property protection for your data.
Cell Types & Donor Options
We use human primary immune cells to ensure translational relevance. The following cell types can be incorporated into your study as scoped.
Peripheral Blood Mononuclear Cells (PBMCs): Fresh or cryopreserved PBMCs from healthy donors (single or pool). Suitable for broad cytokine profiling and early innate activation assessment.
Monocyte-Derived Dendritic Cells (moDCs): CD14+ monocytes differentiated into immature DCs over 5–7 days. Provide a pure DC population for activation marker and sensor engagement studies.
Macrophage Differentiation: Monocytes can be differentiated into M0, M1, or M2 macrophages upon request for evaluating cell-type-specific responses.
Donor Pooling & Diversity: We can pool PBMCs from multiple donors to capture population-level variability, or perform individual donor analysis to assess genetic impact.
Contact Us
Whether you are screening carriers, refining lead candidates, or building a regulatory package, the scientific team at Creative Biogene can help you design the right experimental strategy—from formulation and assay selection to data interpretation. Reach out to discuss a tailored solution for your next project.
