Potency Evaluation Service of Nucleic Acid
Potency is a measure of a product's inherent activity and is the most important quality attribute (CQA) for any complex therapeutic agent, especially nucleic acid drugs. For gene therapies, including plasmid DNA, mRNA, antisense oligonucleotides (ASO), siRNA, and AAV/lentiviral vectors, potency is a key link between product characteristics and clinical efficacy. It is essential for elucidating the mechanism of action (MOA), ensuring batch-to-batch consistency, and guaranteeing long-term product stability. Creative Biolabs offers comprehensive, fully customizable, and regulatory-compliant potency assay solutions designed to provide all-around support for your nucleic acid drug development.
Key Challenges in Potency Assay Development
Due to the complexity of the biological mechanisms underlying nucleic acid therapies, developing robust potency assays presents several challenges:
- High Specificity and Sensitivity Required: Assays must accurately reflect complex, multi-step biological mechanisms (e.g., cell entry, uncoating, transcription, translation, or gene silencing), not just physical properties.
- Regulatory Compliance Demands: Global health regulatory agencies require potency assays to accurately quantify the intended biological activity, necessitating extensive validation under stringent guidelines.
- Model System Development: Establishing stable, reliable, and high-throughput cell lines or relevant primary cell/animal models is crucial for assay implementation but typically requires significant resources.
- High Dimensionality: Potency assays for novel nucleic acid therapies often require measuring multiple endpoints (e.g., target expression and downstream functional output).
Evaluation Background & Industry Insight
A. Nucleic Acid Therapy: A New Frontier in Quality Control
The rapid development of nucleic acid drugs has placed unprecedented demands on product quality and analytical rigor. The complex structure and inherent instability of nucleic acids pose a significant challenge, as their physical quality often cannot be directly correlated with biological function. This necessitates complex potency assessments that go beyond simple concentration measurements.
Figure 1. Schematic illustrating different classes of RNA therapeutics. ASO, antisense oligonucleotide; RNA, ribonucleic acid; RNAi, RNA interference; siRNA, small interfering RNA; miRNA, microRNA; mRNA, messenger RNA; A, adenosine molecule; AAAAA, poly A tail.1
B. Key Difference: Concentration vs. Potency
- Concentration: Refers to the physical quantity or mass of a nucleic acid molecule, typically determined by ultraviolet spectrophotometry or fluorescent dye methods.
- Potency (Activity): Measures the level of biological activity—the intrinsic ability of a nucleic acid to exert its intended therapeutic effect in vitro or in vivo.
- Core Relationship: An effective nucleic acid product must simultaneously possess high concentration and high biological activity.
What is Nucleic Acid Concentration Potency Evaluation?
Our service is a comprehensive analytical solution that combines precise physical concentration measurements with reliable bioactivity assessment (potency). This dual assessment ensures batch-to-batch consistency of your therapeutic products. Since bioactivity cannot be predicted solely by physical concentration, potency assessment is an essential tool for quality control and batch release.
Why Need Potency Evaluation Service of Nucleic Acid?
Nucleic acid therapy technologies play a vital role in modern biomedicine. They can be broadly categorized into three classes: nucleic acids used for gene knockdown, such as small interfering RNA (siRNA), antisense oligonucleotides (ASO), and CRISPR/Cas9 guide RNA (sgRNA); nucleic acids used for gene overexpression (e.g., mRNA and circular RNA); and nucleic acids used for diagnosis, such as probes. The potency of nucleic acids is crucial to their therapeutic efficacy, encompassing their functional activity under different conditions. Methods for detecting the potency of these types of nucleic acids vary. Assessing their in vitro and in vivo potency requires tailored strategies based on their specific functions.
Core Services at Creative Biolabs
Our services span the entire lifecycle of potency assessment, ensuring that you have a consistent and reliable measure of product performance at every stage of development.
Potency Evaluation in Gene Knockdown Nucleic Acids
Nucleic acids such as small interfering RNA (siRNA), antisense oligonucleotides (ASOs), and single guide RNA are used for gene knockdown, which involves reducing target gene or protein levels.
01 In Vitro Potency Assessment
Evaluating the potency of gene knockdown nucleic acids in vitro is crucial for understanding their effectiveness in silencing target genes or proteins. Tab.1 shows various methods used to measure the potency of gene knockdown nucleic acids in vitro.
Tab.1 Methods for determining the potency of gene knockdown nucleic acids in vitro.
| Items | Description | Methods |
|---|---|---|
| Structural Integrity and Stability | Including secondary structures and duplex formation. | Gel Electrophoresis, UV Spectrophotometry, Capillary Electrophoresis |
| Off-Target Analysis | To predict potential off-target sites using Bioinformatics Tools and evaluate potential off-target effects. | TargetScan, Microarray, RNA-seq, Co-Immunoprecipitation (Co-IP), and Mass Spectrometry (MS). |
| mRNA levels | Measures reduction in target mRNA levels. | qRT-PCR |
| Protein levels | Assesses a decrease in target protein levels. | Western blotting, Flow Cytometry |
| Efficiency | Knockdown effect for siRNA | Luciferase Reporter Assay |
| ASO-induced mRNA degradation | Luciferase Reporter Assay | |
| Cleavage efficiency for sgRNA | T7E1 Assay |
02 In vivo Biological Activity
The methods for detecting in vivo potency of nucleic acid drugs vary depending on the disease type. For cancer, the focus is on tumor growth inhibition, imaging, and molecular and cellular changes. In neurological diseases, key assessments include behavioral testing, neuroimaging, and electrophysiological testing. For cardiovascular diseases, the evaluation centers on changes in cardiac function, hemodynamics, imaging techniques, and biomarkers. By employing these diverse methods, the potency and therapeutic effects of nucleic acid drugs can be comprehensively evaluated.
Potency Evaluation in Gene Overexpression Nucleic Acids
Gene overexpression nucleic acids, including mRNA, circRNA, and probes, have diverse applications in therapy and research. They are used in vaccines, direct protein replacement therapies, and as essential tools for genetic studies. Evaluating their potency both in vitro and in vivo is crucial for ensuring their efficacy and safety.
01 In Vitro Potency Assessment
For overexpression use, such as mRNA and circRNA, potency analysis includes qPCR to measure RNA levels, confirming successful transfection and expression, and Western blotting to detect the presence and quantity of the target protein. Functional assays assess the biological activity of the expressed protein, such as enzyme activity or cell viability.
02 In vivo Biological Activity
For mRNA vaccines and therapeutics, immunogenicity assays measure the immune response elicited by mRNA vaccines in animal models or clinical trials.
For circRNA therapeutics, in vivo, expression analysis includes qPCR and RNA-seq to quantify circRNA levels and examine expression profiles in tissues, as well as protein assays to measure the levels and activity of proteins encoded by circRNA. Functional and toxicity studies evaluate the therapeutic effects and potential toxicities in animal models.
Potency Evaluation in Diagnostics Nucleic Acids
Nucleic acids used for diagnostics, such as probes undergo distinct evaluation approaches for both in vitro potency assessment and in vivo imaging and diagnostics. In vitro, hybridization techniques such as Fluorescence in Situ Hybridization (FISH) detect and localize specific nucleic acids within cells, ELISA quantifies nucleic acids captured by probes, and microscopy visualizes probe binding in cellular contexts. For in vivo applications, biodistribution studies analyze the distribution of labeled probes within organisms, adapted FISH techniques detect nucleic acids in live tissues, and advanced imaging modalities like PET and MRI provide real-time visualization of probe localization, crucial for diagnostic and therapeutic purposes.
Platform Technologies Powering Potency
Concentration Measurement Technology
- UV-Vis Spectrophotometry: A rapid and reliable quantitative method, best suited for high-concentration samples.
- Fluorescent Dye Quantification: Capable of distinguishing between dsDNA, single-stranded DNA (ssDNA), and RNA.
- Capillary Electrophoresis (CE): Simultaneously determines purity and concentration, providing high-resolution data.
Core Technologies for Titer/Activity Evaluation
- qPCR/RT-qPCR: The gold standard for determining viral vector titers (AAV/Lenti GC/VC).
- Cell-Based Functional Analysis: Detects target cell uptake using flow cytometry (FACS).
- Functional protein expression: Quantitative analysis was performed using highly sensitive methods.
Target Products and Application Areas
We provide support for quality control needs across the entire nucleic acid development process:
- Nucleic Acid Drugs: mRNA vaccines/therapies, small interfering RNA (siRNA), antisense oligonucleotides (ASO), and plasmid DNA (pDNA).
- Viral Vectors: Quantification of genome copy number (GC/VC) for adeno-associated virus (AAV) and lentivirus.
- Cell and Gene Therapy (CGT): Assessment of cell transfection/transduction efficiency and downstream gene expression levels.
- Quality Control (QC) and Batch Release: Intermediate product testing and final product batch release testing throughout the entire manufacturing process.
Why Choose Our Services?
Unmatched Expertise and Experience
Creative Biolabs is a dedicated partner in gene and cell therapy. Our scientific teams possess deep, specialized knowledge in the development, manufacture, and quality control of advanced nucleic acid therapeutics. This focus ensures that your potency assay is not just a test, but an integral part of your product's clinical narrative.
Stringent Regulatory Compliance
Our laboratory operations adhere strictly to global regulatory quality standards, ensuring all data generated is suitable for direct submission to regulatory authorities worldwide. Every step, from cell culture to data analysis, is governed by rigorously maintained and controlled SOPs, guaranteeing consistency and traceability across projects and batches.
Fully Customizable and Flexible Solutions
We recognize that no two nucleic acid drugs are exactly alike. Our strength lies in our ability to develop bespoke analytical solutions for therapeutics with non-traditional or highly complex mechanisms of action. Whether you require an assay for a novel fusion protein expressed by an mRNA or a multi-modal gene therapy, we adapt our platforms and expertise to your specific needs.
Robust Quality Assurance (QA/QC)
Quality is embedded in every project. Our dedicated Quality Assurance unit oversees all operations, providing an independent check to ensure compliance and data integrity. We utilize a comprehensive, validated document management system to ensure all raw data, analysis files, and audit trails are complete, secure, and fully traceable.
Our Collaborative Process
We structure our partnership into four transparent phases to maximize efficiency and ensure alignment with your strategic goals.
-
Phase 1
Consultation and Strategic Planning
The process begins with an in-depth consultation to understand your drug product's characteristics, target indication, and precise MOA.
- Goal Definition: Defining the regulatory purpose (release, stability, characterization) and the required sensitivity of the potency assay.
- Feasibility Assessment: Reviewing existing data (if any) and selecting the most appropriate biological system (cell line, primary cells) and technical readout.
- Project Proposal: Delivering a detailed plan outlining the assay format, required reagents, timelines, and deliverables.
-
Phase 2
Assay Development and Optimization
Our scientists execute the laboratory work to translate the concept into a functional assay.
- Proof-of-Concept: Establishing the biological response window and preliminary assay conditions.
- Optimization: Systematically fine-tuning concentration ranges, incubation conditions, and signal detection methods to achieve optimal signal-to-noise ratio and dose-response curve characteristics.
- Establishment of Acceptance Criteria: Defining the initial system suitability and assay performance metrics.
-
Phase 3
Validation and Execution
Once optimized, the assay moves into the formal validation and testing phase.
- QC Testing: Applying the validated assay to test drug substance and drug product lots for release and stability studies.
- Troubleshooting & Transfer: Providing comprehensive support for the eventual transfer of the validated method to your internal QC lab or a third-party manufacturer, including on-site training and technical documentation.
-
Phase 4
Reporting and Data Analysis
We provide meticulously detailed reports that transform complex data into actionable insights.
- Raw Data Compilation: All raw data files from analytical instruments are compiled and provided.
- Statistical Analysis: All potency calculations, including EC50 values and relative potency (RP) determination, are performed using validated statistical software, compliant with global regulatory standards for biological assays.
Result Delivery
Our commitment extends beyond generating data; we deliver comprehensive, submission-ready documentation that accelerates your regulatory timelines.
- Final Study Report: A detailed, auditable document summarizing the study rationale, methods, raw data, statistical analysis, conclusions, and any observed deviations. This report is authored by senior scientists and reviewed by our QA team.
- Data Package: Providing a complete electronic data package, including all instrument printouts, spreadsheet calculations, dose-response curves, and the 4PL/5PL regression models used for potency calculation. Data is presented in formats suitable for direct incorporation into regulatory dossiers.
- Method SOP: A rigorously documented, step-by-step Standard Operating Procedure for the final validated assay, ensuring consistency and ease of transfer for future testing.
Frequently Asked Questions
Q: What is the main difference between titer assays and potency assays for AAV vectors?
A: Titer assays (e.g., genomic titer determination via qPCR) measures the physical quantity of the vector genome. Potency assays measure the vector's ability to transduce cells and express therapeutic genes. Regulatory agencies require both assays, but potency assays are considered a critical critical quality attribute (CQA) that demonstrates biological activity and predicts in vivo efficacy.
Q: Can you develop potency assays for novel mRNA therapies targeting intracellular proteins?
A: Yes. We focus on non-traditional targets. For intracellular proteins, we typically develop cell-based assays using techniques such as Western blotting or high-content screening (HCS), employing sensitive, specific antibodies to measure post-translational protein levels, and often combining this with downstream functional assays (e.g., phosphorylation status or pathway activation) to reveal the complete mechanism of action (MOA).
Q: How long does the typical potency assay development and validation process take?
A: The timeline depends heavily on the complexity of the drug's mechanism of action and the availability of critical reagents. Typically, the development and optimization phase takes about 8-12 weeks, while the complete ICH validation phase requires an additional 6-10 weeks. We work closely with clients to develop accelerated timelines if needed.
Q: Does your service include stability indicator potency testing?
A: Absolutely. Validation is crucial in demonstrating the stability indicator capability of the assay method. We ensure that the assay method can detect and quantify the loss of activity after forced degradation (e.g., heat stress or repeated freeze-thaw cycles), a critical requirement for determining product shelf life and storage conditions.
Q: If my nucleic acid drug has a complex, multifunctional mechanism of action, how should potency testing be performed?
A: For multifunctional drugs, a single potency assay may not be sufficient. We employ a multiplex assay strategy, where a primary assay measures the most critical functional steps (e.g., target binding), and a secondary assay measures other important biological functions. The final potency indicator is determined by combining the results of these validated assays, ensuring an assessment of all aspects of drug activity.
Contact Us Today
- Ready to move forward? Fill out our project inquiry form or contact us directly to schedule a confidential discussion about your specific potency evaluation needs.
- Secure your submission success with scientifically rigorous and regulatory-compliant potency data.
Reference
- Damase T R, Sukhovershin R, Boada C, et al. The limitless future of RNA therapeutics. Frontiers in bioengineering and biotechnology, 2021, 9: 628137. https://doi.org/10.3389/fbioe.2021.628137 (Distributed under Open Access license CC BY 4.0, without modification.)
