Sensitive and specific LC-MS/MS quantification of antisense oligonucleotides, siRNA, mRNA, aptamers, and conjugated oligonucleotides.
Oligonucleotide drug bioanalysis is the quantification and characterization of synthetic DNA- or RNA-based therapeutics in biological matrices to support pharmacokinetic (PK), toxicokinetic (TK), and biodistribution studies. These analyses enable accurate exposure-response assessment for antisense oligonucleotides (ASOs), small interfering RNA (siRNA), messenger RNA (mRNA), aptamers, guide RNA (gRNA), and conjugated oligonucleotide therapeutics (e.g., ARCs, AOCs).
Our bioanalytical services employ validated LC–MS/MS and hybrid LC–MS/MS platforms to measure intact oligonucleotides and their metabolites in plasma, tissue, and other matrices. Method development optimizes extraction efficiency, ionization, and chromatographic separation to achieve the sensitivity and selectivity required for early- to late-stage development programs.
From discovery through clinical development, we deliver GLP/GCLP-compliant bioanalysis that supports IND/NDA submissions. Each project includes a dedicated study scientist, milestone review calls, and a final report with PK/TK parameters and any relevant metabolite profiling.
Key Features
LC–MS/MS and Hybrid LC–MS/MS Method Development
We develop robust LC–MS/MS and hybrid LC–MS/MS (immunoaffinity capture coupled with LC–MS/MS) methods for the quantification of oligonucleotide therapeutics in complex biological matrices. Method optimization includes ion-pairing reagent selection, column chemistry, and mass spectrometry parameters to achieve low ng/mL sensitivity and specificity for the full-length oligonucleotide and its metabolites.
GLP/GCLP Method Validation
Validation is performed in accordance with FDA and EMA bioanalytical method validation guidance. Parameters include accuracy, precision, selectivity, matrix effect, recovery, stability (benchtop, freeze-thaw, long-term), and dilution integrity. For hybrid LC–MS/MS methods, additional characterization of the capture probe and elution conditions is conducted to ensure robust performance.
Sample Extraction and Analysis
Our extraction workflows employ solid-phase extraction (SPE), liquid-liquid extraction (LLE), or protein precipitation, optimized per analyte class. High-resolution mass spectrometry (HRMS) and triple quadrupole (QqQ) platforms are used for quantitative and qualitative analysis. We routinely analyze plasma, serum, tissue homogenates, CSF, and urine.
Metabolite Identification and PK/TK Parameter Calculation
Using HRMS and data-dependent acquisition, we can identify and profile oligonucleotide metabolites (e.g., chain-shortened, oxidized, nuclease degradation products). Combined with quantitative analysis, we calculate standard PK/TK parameters (Cmax, AUC, t½, CL/F, Vz/F) and provide a comprehensive study report suitable for regulatory submission.
Modalities Supported
Our bioanalysis platform accommodates a broad range of oligonucleotide therapeutic modalities, each with distinct bioanalytical considerations. The following modalities are routinely analyzed in our laboratory:
Antisense Oligonucleotides (ASOs): Single-stranded DNA-like oligomers that modulate gene expression via RNase H recruitment or steric block. Bioanalysis requires intact ASO and metabolite quantification, often in plasma and liver tissue.
Small Interfering RNA (siRNA): Double-stranded RNA triggers of RNA interference. Quantification of both sense and antisense strands, including strand-specific analysis after chemical modifications for stability.
Messenger RNA (mRNA): Large, modified mRNA constructs for protein replacement or vaccine applications. Bioanalysis addresses intact mRNA quantitation, fragmentation, and biodistribution using LC–MS/MS and molecular methods.
Aptamers and Guide RNA (gRNA): Single-stranded RNA or DNA aptamers and CRISPR-associated gRNA. LC–MS/MS methods are adapted for secondary structure stability and ionization efficiency.
Conjugated Oligonucleotide Therapies (ARCs, AOCs): Oligonucleotides conjugated to targeting moieties (e.g., GalNAc, antibodies) for enhanced delivery. Bioanalysis covers the conjugated molecule and released oligonucleotide, including tissue distribution and linker stability.
Workflow
Each oligonucleotide bioanalysis project follows a structured, phase-appropriate process from initial consultation to final report. Workflows are adapted to the specific modality, matrix, and regulatory stage.
Scientific Consultation and Study Design: A senior scientist reviews the molecular properties, target matrix, expected concentration range, and regulatory requirements. A project plan is proposed, including method development approach, validation parameters, and sample analysis timeline.
Method Development and Optimization: Extraction conditions (SPE, LLE, precipitation), chromatographic separation (ion-pairing RP-HPLC or HILIC), and MS parameters (ionization mode, MRM transitions, HRMS settings) are optimized using reference standards and control matrix.
Method Validation: Full GLP/GCLP validation according to ICH M10 or FDA guidance. Acceptance criteria are met before proceeding to sample analysis. Stability assessments cover bench-top, freeze-thaw, and long-term storage.
Sample Analysis and Data Reporting: Study samples are analyzed in batches with calibration curves, QCs, and blanks. Data are processed with validated software, and PK/TK parameters are calculated. A final report is generated, including method description, raw data, and interpretation.
Regulatory Support and Submission Documentation: We provide documentation suitable for IND/NDA submissions, including validation reports, sample analysis reports, and responses to regulatory queries. Our scientific team can assist in presenting bioanalytical data in a regulatory context.
Regulatory Experience
Our bioanalytical team has supported multiple oligonucleotide programs through IND-enabling studies and clinical development. We understand the regulatory expectations for bioanalysis of modified oligonucleotides and can advise on study design to meet FDA and EMA requirements.
Quality Assurance
All oligonucleotide bioanalysis services are performed under a GLP/GCLP quality system that complies with FDA 21 CFR Part 58 and OECD Principles of GLP. Studies are conducted by qualified scientists with documented training, and all data are generated on validated instruments with audit trail capabilities.
Each project is assigned a dedicated study director or principal investigator who oversees method development, validation, and sample analysis. Reports are issued with full raw data, chromatograms, and statistical summaries. We welcome sponsor audits and regulatory inspections.
Why Choose Us
Deep Modality Expertise: Our scientists have years of experience with ASOs, siRNA, mRNA, aptamers, and conjugated oligonucleotides. We understand the unique bioanalytical challenges of each modality.
Platform Versatility: We employ both triple quadrupole and high-resolution mass spectrometry, as well as hybrid LC-MS/MS, to deliver the sensitivity and selectivity required for intact oligonucleotide and metabolite quantification.
Regulatory Track Record: We have supported multiple oligonucleotide programs through successful IND submissions and clinical development. Our documentation is structured for straightforward regulatory review.
Collaborative Scientific Approach: From study design to report delivery, you work directly with PhD-level scientists who understand your program goals and can adapt methods to evolving project needs.
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.
Biological Matrices and Sample Types
Our validated extraction and LC-MS/MS methods are suitable for a wide range of biological matrices commonly used in oligonucleotide drug development. Sample stability, matrix effect, and recovery are assessed for each new matrix-modality combination.
Plasma and Serum: Standard matrices for PK/TK analysis. We optimize extraction to minimize matrix interference from proteins and lipids.
Tissue Homogenates (Liver, Kidney, Spleen, Lung): Key for biodistribution studies. Tissue lysis and homogenization protocols are tailored to the oligonucleotide chemistry and tissue type.
Cerebrospinal Fluid (CSF): Relevant for CNS-targeted oligonucleotides. Low-volume, high-sensitivity methods required; our methods achieve sub-nanogram per mL quantification.
Urine and Bile: Used to assess renal and hepatic elimination pathways. Extraction methods account for high salt and variable pH.
Contact Us
Whether you are initiating a PK/TK study, validating a bioanalytical method, or preparing an IND-enabling data package, the scientific team at Creative Biogene can help you design the right bioanalysis strategy—from method development through regulatory submission support. Reach out to discuss a tailored solution for your next project.
