Purity Determination Service of Nucleic Acid
In the highly regulated field of nucleic acid therapy, purity is not an abstract concept, but a fundamental guarantee of safety and efficacy. Creative Biolabs combines cutting-edge technology with deep scientific expertise to provide you with reliable nucleic acid analysis services. We offer crucial data and insights to help you reduce R&D risks, meet research requirements, and ultimately bring transformative nucleic acid drugs to market faster and with greater confidence.
Nucleic Acid Therapy Introduction
The emergence of nucleic acid therapy has fundamentally reshaped the landscape of drug innovation. These molecules are diverse, ranging from short-chain interfering oligonucleotides (siRNA), antisense sequences (ASO), and messenger RNA (mRNA) to complex plasmid DNA (pDNA) and viral vectors, representing a paradigm shift distinct from traditional small-molecule chemistry and protein biologics. Their mechanisms of action are not to block active sites or stimulate receptors, but rather to directly regulate gene expression—silencing disease-causing genes, editing genome sequences, or directing cellular mechanisms to produce therapeutic proteins.
Figure 1. Overview of types, modes of action, and delivery strategies of nucleic acid-based therapeutics for anti-cancer therapy. siRNA, small interfering RNA; miR, microRNA; ASO, antisense oligonucleotide; CNA, catalytic nucleic acid; gRNA, guide RNA; pDNA, plasmid DNA; mRNA, messenger RNA.1
The Golden Age of Nucleic Acid Drugs
We are currently witnessing a "golden age" of nucleic acid drugs. The rapid and successful development of mRNA-based vaccines has demonstrated the feasibility and speed of nucleic acid technology application globally. This success has spurred accelerated research into all treatment modalities:
- Antisense oligonucleotides (ASOs): These single-stranded molecules are designed to bind to complementary mRNA sequences, thereby inhibiting protein translation or altering the splicing of precursor mRNA.
- Small interfering RNA (siRNA): siRNAs function through RNA-induced silencing complexes (RISCs), promoting the degradation of target mRNA, thus achieving effective gene knockdown.
- Aptamers: As "chemical antibodies," these nucleic acid sequences can fold into complex three-dimensional structures, thereby binding to target proteins or small molecules with high affinity and specificity.
- Viral and non-viral vectors: Nucleic acid payloads in plasmid DNA (pDNA) and viral delivery systems (e.g., adeno-associated virus, lentivirus) are crucial components of gene and cell therapies, representing the forefront of regenerative medicine.
Evaluation Background & Industry Insight of Nucleic Acid Drug
Process-Related Impurities
These are residual substances derived from the manufacturing process.
- Residual Solvents and Reagents: Unreacted linkers, protecting groups, and organic solvents used during solid-phase synthesis.
- Counterions: The presence of non-target metal ions or counterions required for formulation stability, which must be accurately quantified.
- Endotoxins and Bioburden: Critical contaminants for injectable products, requiring stringent control and release testing.
The Purity-Function Relationship
Even minor variations in nucleic acid purity can have catastrophic consequences for clinical outcomes. Impurities can:
- Reduce Potency: Truncated or modified sequences may lack the intended binding or gene-silencing activity, leading to under-dosing.
- Increase Immunogenicity: Double-stranded RNA contaminants in $\text{mRNA}$ preparations can activate toll-like receptors (TLRs), triggering innate immune responses that cause inflammation and rapid clearance of the therapeutic agent.
- Affect Stability: Impurities may catalyze degradation pathways, compromising the shelf-life and efficacy of the final drug product.
Purity Determination Methods of Nucleic Acid
Robust quality assurance hinges on a portfolio of complementary analytical techniques, each designed to interrogate a specific aspect of molecular integrity and composition.
Anion-Exchange Chromatography
It offers excellent resolution for length variants, which is crucial for the synthesis of oligonucleotides.
Reversed-Phase HPLC
This method is based on hydrophobic separation of nucleic acids, thus enabling efficient separation of subtle differences in chemical modifications.
Capillary Electrophoresis
It is a powerful tool for analyzing the integrity and size distribution of larger species such as mRNA and plasmid DNA.
Intact Mass Analysis (HRMS)
High-resolution mass spectrometry determines the precise mass of intact nucleic acid molecules. This confirms the sequence and all anticipated chemical modifications.
UV Spectrophotometry
A260 measurement is the standard method for quantifying nucleic acid concentrations.
Endotoxin Testing
A critical test for process-related impurities, confirming the absence of bacterial endotoxins to meet strict regulatory requirements for injectable drug products.
Our Services
At Creative Biolabs, our Purity Determination Service for Nucleic Acids is designed to provide comprehensive analytical packages that span the entire product life cycle—from early discovery to cGMP-compliant final product release. We offer a phased approach to quality control, tailored to the specific modality (ASO, $\text{siRNA}$, $\text{mRNA}$, $\text{pDNA}$, or vector).
The nucleic acid impurity test methods.
| Items | Description | Test Methods |
|---|---|---|
| Synthetic impurities | These impurities include deletion sequences ('shortmers'), addition sequences ('longmers'), modified full-length species, End-modification impurities, and Modification impurities. | High-Performance Liquid Chromatography (HPLC), capillary electrophoresis (CE), or mass spectrometry |
| Ions and salts | Ensure that the concentrations of ions and salts in solvents, reagents, and buffers used in the synthesis reaction meet specifications. | Ion chromatography (IC) or atomic absorption spectroscopy (AAS). |
| Solvent residues | Detect solvent residues used during synthesis to ensure the product does not contain them. | Gas chromatography (GC) and liquid chromatography (LC). |
| Exogenous DNA contamination | Detecting the presence of exogenous DNA or RNA contamination, originating from the laboratory environment, instruments, reagents, or operators, is crucial when synthetic oligos are employed for diverse applications. | Polymerase chain reaction (PCR) or colorimetric assays. |
| Exogenous RNA contamination | Reverse Transcription Polymerase Chain Reaction (RT-PCR) or Southern Blot Assays. | |
| Exogenous Protein contamination | The primary routes of external protein contamination during nucleic acid production involve the introduction of enzymes (such as nucleases and proteases) and the presence of residual intracellular proteins. | Nano Orange Assay. |
Key Technology Platform Highlights
Our proprietary analytical workflows integrate standard industry tools with custom-developed methodologies for handling novel nucleic acid constructs.
- Integrated Chromatographic-Mass Spectrometric Workflows
- Advanced mRNA Purity Assessment
- Oligonucleotide Isomer Resolution
Partnership and Success Stories
Creative Biolabs acts as a strategic analytical partner, not just a service provider. Our engagements are defined by deep collaboration, applying our PhD-level expertise to your most challenging analytical problems.
Case Study 01: Accelerated ASO Development: We partnered with a mid-sized biotechnology company developing a novel ASO therapy. The client encountered low concentrations of N-1 impurities during efficacy studies. Our team rapidly implemented a highly selective AEC method that improved baseline resolution by 40%, enabling the manufacturer to optimize its purification process.
Case Study 02: Vector Payload Integrity: We developed a qPCR-based assay for a gene therapy client, combined with gel electrophoresis integrity testing, to accurately quantify genomic titers and assess the structural integrity of pDNA payloads in their viral vectors. This rigorous quality control provided the necessary documentation to meet stringent regulatory requirements for product release.
(Note: All names, products, and specific numerical data are conceptual to avoid infringement and to illustrate our scope of work.)
Our Collaborative Process
We follow a transparent and iterative process to ensure the analytical workflow perfectly aligns with your specific therapeutic and regulatory goals.
Consultation and Scope Definition
An initial meeting with our principal scientists to define the therapeutic modality, the stage of development, the target purity profile, and all relevant CQAs.
Method Development and Feasibility
Based on the molecular profile, we select the most appropriate orthogonal methods, perform feasibility testing, and establish preliminary method parameters.
Method Validation and Transfer
For later-stage products, we execute a full analytical method validation according to standards. We ensure seamless method transfer to your internal QC laboratories if required.
High-throughput analysis of your API or drug product.
Data Interpretation and Review
Our team provides a written analytical report, interpreting the results in the context of your manufacturing process and regulatory strategy, going beyond simple raw data delivery.
Frequently Asked Questions
Q: What is the minimum sample size required for a full suite of purity analyses?
A: Sample size largely depends on the molecular weight and complexity of the nucleic acid. Typically, a full suite of identification and purity assays for oligonucleotides requires 1-5 mg of sample, but for high-titer carrier formulations, even less is needed. We will conduct a feasibility assessment based on the sample size provided by the client.
Q: How do you handle novel chemical modifications that may not have standard HPLC methods?
A: We employ mass spectrometry-guided purification and analysis methods. By coupling HPLC with HRMS, we can rapidly determine optimal separation conditions based on the quality of the components, without relying solely on nonspecific UV detection, thus enabling rapid adaptation to new chemical modifications.
Q: What is the typical turnaround time for standard oligonucleotide purity assays?
A: For routine pre-validation assays, it is typically completed within 5-7 business days after receiving the sample. For complex method development, the timeline will be determined collaboratively based on the project scope.
Q: Can you distinguish between isomer impurities (e.g., diastereomers of thiophosphates)?
A: While standard purity analysis methods like capillary electrophoresis (CE) and reversed-phase high-performance liquid chromatography (IP-RP-HPLC) can sometimes separate diastereomers, precise analysis requires specialized chiral separation methods or nuclear magnetic resonance (NMR). We can arrange these analyses through our partner network.
Q: How do you quantify double-stranded RNA (dsRNA) impurities in mRNA samples?
A: We employ highly specific and sensitive methods to distinguish and quantify double-stranded RNA impurities from single-stranded mRNA products, providing critical quality attributes for your stability and safety studies.
Connect with Us Anytime!
The purity of nucleic acids is essential for ensuring their effectiveness in a wide range of applications. Choosing appropriate sample preparation methods and purity detection techniques, as well as rigorously controlling impurity levels, are of paramount importance for ensuring the accuracy and reliability of experimental results. Creative Biolabs is committed to offering high-quality purity test services for worldwide customers. For more detailed information, please feel free to contact us and get a quote. We'll reach out to you within 24 hours and formulate the best method for your project.
Reference
- Lee M, Lee M, Song Y, et al. Recent advances and prospects of nucleic acid therapeutics for anti-cancer therapy. Molecules, 2024, 29(19): 4737. https://doi.org/10.3390/bios14030139 (Distributed under Open Access license CC BY 4.0, without modification.)
