Mediford Corporation

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Nucleic Acid Drug Analysis

Nucleic Acid Drug Analysis

In recent years, the development of next-generation drugs has been gaining momentum worldwide. Among them, nucleic acid drugs have been continuously launched every year since around 2016, representing a novel modality that is anticipated to see further advancements in the future. In drug development, it is important to evaluate pharmacokinetics, and it is essential to quantify drug concentrations in biological samples.

Under the framework of GLP (Good Laboratory Practice), Mediford Corporation conducts the development and validation of analytical methods, as well as the determination of drug concentrations for nucleic acid drugs in biological samples such as plasma and tissue. We also carry out metabolite screening studies for nucleic acid drugs in accordance with reliability standards.

Service Details

Drug concentration determination in biological samples

LC-MS/MS method for nucleic acid drug analysis

We offer contract services such as method development, method validation, and drug concentration determination. Compared to the hybridization-ECLIA (electrochemiluminescence immunoassay) method mentioned later, the LC-MS/MS (liquid chromatography-tandem mass spectrometry) method offers higher selectivity, making it suitable for metabolite determination of nucleic acid drugs. The versatility of analytical conditions and the ability to directly detect metabolites highlight the advantages of the LC-MS/MS method. Additionally, since the synthesis of probes required for hybridization assays is not necessary, the LC-MS/MS method is well-suited for situations where rapid method development and measurement are required, such as in the discovery stage of candidate compounds for drug development.

Case study of analyzing antisense (nucleic acid drug) [sample: 50 microliters of plasma]
LC-MS/MS (Triple Quad 5500)
Calibration curve
Analyte: Nusinersen
Range: 0.2-100 ng/mL in plasma
Nexera X2 (Shimadzu) / Triple Quad 5500 (Sciex)

Hybridization-ECLIA method for nucleic acid drug analysis

We provide contract services including probe design and arrangement, labeling of detection reagents (e.g., Ruthenium), method development, method validation, and drug concentration determination. The hybridization-ECLIA (electrochemiluminescence immunoassay) method requires simple sample preparation and allows for high-throughput measurement, making it effective for analyzing numerous samples. It offers higher sensitivity compared to LC-MS/MS, enabling quantification with small sample volumes.

Case study of analyzing antisense (nucleic acid drug) [sample: 1.25 microliters of plasma]
Hybridization-ECL (MESO QuickPlex SQ120)
Calibration curve
Analyte: Nusinersen
Range: 0.7-713 ng/mL in plasma
MESO QuickPlex SQ120
(Meso Scale Diagnostics, LLC)

Drug concentration determination results by analytical method

In a study where nusinersen was administered in a single intravenous dose (1mg/kg) to Crl:CD(SD) rats, the quantification of drug concentrations in actual samples showed a good correlation between the two analytical methods, LC-MS/MS and Hybridization-ECLIA.

Comparison of concentrations of Nusinersen between two quantification methods,
LC-MS/MS (Triple Quad 5500) and Hybridization-ECLIA (MESO QuickPlex SQ120)
Administration: Crl:CD(SD), intravenous, 1 mg/kg, single dosing

Metabolite screening for nucleic acid drugs

Typically, drugs ingested into the body are metabolized into structures that are easily excreted, but active metabolites are sometimes produced. Therefore, it is crucial to quantify the concentrations of drugs and their metabolites in biological samples for the safety assessment of drugs. Japanese guidelines*1 state that for nucleic acid drugs, non-clinical safety assessment is necessary for metabolites that contains chemically modified parts. Mediford Corporation conducts metabolite screening services for nucleic acid drugs (cold/hot) using LC-MS (Orbitrap) under reliability standards.

*1: ICH-M3(R2)

Instrument  
LC Nexera X2 System (Shimadzu)
MS/MS (Orbitrap) Q Exactive Focus (Thermo Fisher Scientific)
Maximum Resolution: 70,000@m/z=200
Mass error < ±3 ppm (external standard). < ±1 ppm (internal standard)
Software Xcalibur 4.1. Compound Discoverer 2.1 (Thermo Fisher Scientific)

Case studies of metabolite screening

Metabolites of nucleic acid pharmaceuticals in rat serum after 72 hours of incubation at 37℃
LNA-A: 5-CTAgttcacgaaTGC-3
Upper case: LNA, lower case: DNA, C: 5-C, all phosphorothioate backbone
Metabolite estimation in rat serum after 72 hours incubation at 37℃

Simultaneous quantification of unchanged nucleic acid drugs and their metabolites

Using the LC-MS/MS method, we can simultaneously quantify both the unchanged form and metabolites of nucleic acid drugs.

Simultaneous quantification of Nusinersen and metabolites (3’N-1, 3’N-2)
by LC-MS/MS（QTRAP 6500+）
Administration: Crl:CD(SD), intravenous, 1 mg/kg, single dosing