Observations from a decade of oligonucleotide bioanalysis by LC-MS.

There is a growing need for efficient bioanalysis of oligonucleotide therapeutics. This broad class of molecules presents numerous challenges relative to traditional small molecule therapeutics. Methodologies including ligand-binding assays or polymerase chain reaction may be fit-for-purpose in many instances, but liquid chromatography coupled to mass spectrometry (LC-MS) often delivers the best balance of sensitivity and selectivity. Over the last decade, we have engaged with many such molecules and derived insights into challenges and solutions. Herein, we provide four case studies illustrating challenges we have encountered. These issues include low or variable analyte recovery, poor resolution from related species, chromatographic abnormalities or challenging sensitivity. We present a summary of considerations, based on these experiences, to assist others working in the area.

High-sensitivity workflow for LC-MS-based analysis of GalNAc-conjugated oligonucleotides: a case study.

Aim: Mass-selective quantitation is a powerful attribute of LC-MS as a platform for bioanalysis. Here, a sensitive LC-MS approach has been validated for an oligonucleotide having chemical modifications (e.g., N-acetylgalactosamine [GalNAc] conjugated), to distinguish between the conjugated and unconjugated forms of the oligonucleotide, thereby enabling a nuanced view of the pharmacokinetic profile. Results: A high-sensitivity methodology for mass-specific measurement of AZD8233, a GalNAc-conjugated 16-mer oligonucleotide, using LLE-SPE with optimized LC conditions and detection of a low-mass fragment ion was successfully validated in the range of 0.20-100 ng/ml in human plasma. Conclusion: The AZD8233 LC-MS methodology adds valuable insight on the GalNAc linker's in vivo stability to the program and should be broadly applicable to oligonucleotides requiring high sensitivity and mass-selective measurement for quantitative discrimination from metabolites and endogenous interferences.

Whole blood stability in quantitative bioanalysis.

Establishing stability at all stages of a sample's lifespan is a critical part of performing regulated bioanalysis. For plasma assays, this includes the duration between when blood is drawn and when that blood is centrifuged to produce plasma. Here, we provide a discussion of current regulatory expectations around whole blood stability testing for LC-MS plasma assays, as well as the two primary experimental approaches utilized to assess whole blood stability. Next, we interrogated a large dataset of validated methods (1076 methods, the vast majority of which were for measurement of small molecules) to assess the correlation between whole blood and plasma stability profiles, finding them to be highly correlated. Finally, we summarize unique case studies; we have encountered during WB stability testing which offer lessons that may be broadly applicable.

Determination of osimertinib in human plasma, urine and cerebrospinal fluid.

Aim: Osimertinib (Tagrisso, AZD9291) has been approved for the treatment of patients with metastatic EGFRm T790M non-small-cell lung cancer. Results: Rapid and sensitive LC-MC/MS methods were developed for osimertinib and its metabolites, AZ13597550 and AZ13575104, in human plasma (low- and high-range), urine and cerebrospinal fluid. We discuss the challenges of these multi-analyte and multiple matrix assays. The methods have been successfully validated and used for the analysis of over 20,000 clinical samples, with successful incurred sample reproducibility. Conclusion: The assays have been shown to be selective, accurate and robust, providing high-throughput analysis during the clinical development of osimertinib.

LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

AIM: We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. METHODOLOGY: We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. CONCLUSION: Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites.

BACKGROUND: LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. RESULTS/METHODOLOGY: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5´n-1, 5´n-2, 5´n-3, 3´n-1, 3´n-2 and 3´n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. CONCLUSION: We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3´ and 5´ metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.

Case studies: the impact of nonanalyte components on LC-MS/MS-based bioanalysis: strategies for identifying and overcoming matrix effects.

Achieving sufficient selectivity in bioanalysis is critical to ensure accurate quantitation of drugs and metabolites in biological matrices. Matrix effects most classically refer to modification of ionization efficiency of an analyte in the presence of matrix components. However, nonanalyte or matrix components present in samples can adversely impact the performance of a bioanalytical method and are broadly considered as matrix effects. For the current manuscript, we expand the scope to include matrix elements that contribute to isobaric interference and measurement bias. These three categories of matrix effects are illustrated with real examples encountered. The causes, symptoms, and suggested strategies and resolutions for each form of matrix effects are discussed. Each case is presented in the format of situation/action/result to facilitate reading.

Development of a bioanalytical method for quantification of a 15-mer oligonucleotide at sub-ng/ml concentrations using LC-MS/MS.

LC-MS/MS provides a powerful technique for the selective quantification of therapeutic oligonucleotides; however, the LOQ (typically >1 ng/ml) may be higher than desirable for clinical bioanalysis. A method has been developed to allow quantification of a 15-mer unmodified DNA oligonucleotide in human plasma using SPE and UHPLC with MS/MS detection. The LOQ of this assay was 0.05 nM (∼250 pg/ml). This method was then further developed by the inclusion of online SPE to increase loading and apply additional sample cleanup. This allowed for improved assay precision at lower concentrations and increased signal, thus allowing the method to be validated over the range of 10-4000 pM (approximately 50-20,000 pg/ml). The method is accurate, precise and selective and it provides proof-of-concept for sub-ng/ml, high-throughput quantification of oligonucleotides using online SPE coupled to ion-pair, reversed-phase LC-MS/MS.

Bioanalytical approaches to analyzing peptides and proteins by LC--MS/MS.

Peptides and proteins have been utilized as therapeutic agents for over 40 years. Traditional approaches to quantify these molecules in biological matrices have utilized immunoassay approaches that can be time inefficient, lack assay specificity and have limited analytical ranges. The advances in sample preparation technologies, chromatographic systems and their chemistries, mass spectrometers and their software over the last decade have meant that LC-MS/MS approaches to peptide and protein quantification are feasible and can overcome the problems associated with quantification by immunoassay. In this article we present an overview of the challenges and approaches to overcome them when performing quantitative bioanalysis of peptides and proteins by LC-MS/MS.

Validation of a bioanalytical method for the quantification of a therapeutic peptide, ramoplanin, in human dried blood spots using LC-MS/MS.

A method has been developed and validated for the quantification of ramoplanin, a 2554 Da peptide antibiotic, in human dried blood spots using high-performance liquid chromatography with tandem mass spectrometric detection. The validation data meet FDA acceptance criteria for bioanalytical assays and cover the quantification of ramoplanin over the range 10-5000 ng/mL. The assay determines ramoplanin at the same lower limit of quantification as conventional liquid sample methods. Dried blood spot analysis provides an approach for quantification of peptide therapeutics and delivers significant benefits for sample collection and handling and also sample cleanup over conventional plasma and serum assays.