Bioanalysis最新文献

Aims: Polyethylene glycol (PEG) is used in many applications including drug development. Due to exposure to environmental products, there is a high prevalence of preexisting anti-PEG antibodies in the global human population. The presence of anti-PEG antibodies is a concern for potentially reducing the efficacy of therapeutics after administration and represents a risk of safety events after exposure to PEGylated drug products. We developed and validated a creative and sensitive method for the detection of anti-PEG antibodies in human serum to support clinical programs for PEGylated drugs.

Methods: In this method, biotin-PEG streptavidin beads were used to extract anti-PEG antibodies from human serum for analysis in an anti-PEG ELISA assay. The same serum sample was analyzed in an anti-drug antibody assay.

Results: The anti-PEG antibody assay was validated with a screening cut point of 1.41 normalized signal, confirmatory cut point of 32.2% inhibition, sensitivity of 7.81 ng/mL and sufficient reproducibility, selectivity, and drug tolerance in accordance with the FDA 2019 Immunogenicity guidance.

Conclusion: This method of removal of anti-PEG antibodies enables the use of a single sample to detect anti-drug and anti-PEG antibodies to support drug development programs.

Following up on our most recent discussion paper focusing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e., the what?) and another on the practical considerations (i.e., the how). In this paper, we discuss 'the what'. Both papers should be helpful for the bioanalytical community to better understand the challenges and provide an insight on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or ligand binding assays for biotherapeutics.

The ICH M10 guideline establishes global standards for bioanalytical method validation for pharmacokinetic assays, focusing on data reliability and accuracy across studies. A significant component is cross-validation, which should be performed to ensure data comparability when multiple methods or laboratories are involved in a single study or across studies where comparison will be performed. However, ICH M10 does not specify acceptance criteria for cross-validation, creating challenges for the industry because traditionally many laboratories have always utilized acceptance criteria to "pass" or "fail" the study. This editorial discusses how bioanalytical labs should conduct cross-validation for PK assays post-ICH M10, highlighting the role of statistical methods and the need for close collaboration with clinical pharmacology and biostatistics departments. Proper implementation and strategic focus on relevant studies are essential for effective cross-validation.

Aims: Gastrointestinal stromal tumors (GISTs) account for about 80% of the mesenchymal tumors of the GI tract. About 5000-6000 patients are diagnosed in the United States (US) alone, and up to 14.5 cases per million discovered in Europe annually. Avapritinib (AVP) is a potent selective targeted medication that has been recently approved, by the US Food and Drug Administration, in 2020 for treatment of GISTs. AVP is currently considered the first-line treatment for mutant GIST, which is resistant to other medications. This in turn stimulates the need for fast, green, and efficient methods for routine AVP estimation in quality control and clinical studies.

Materials and methods: The proposed approach designs a spectrofluorimetric tool to estimate AVP in different matrices, based on a nucleophilic substitution reaction. A highly fluorescent product was measured at 535 nm following excitation at 470 nm. The research procedure was bioanalytically validated within AVP range between 80 and 900 ng mL^-1, where the limit of quantitation (LOQ) was 15.78 ng mL^-1.

Conclusion: The developed approach was successfully applied to investigate AVP in content uniformity testing of tablet dosage forms, and biological plasma in AVP pharmacokinetic study. The proposed approach could be recommended for AVP therapeutic drug monitoring.

Introduction: Selecting the optimal platforms to quantitate cytokines is challenging due to varying performance and the plethora of options available.

Aims: To compare performance of three highly sensitive, multiplex assays on three different platforms - MSD S-plex, Olink Target 48, and Quanterix SP-X - to MSD V-plex which is widely used for quantitative cytokine assay.

Methods: Serum and stimulated plasma samples were analyzed across each platform. The proportion of quantifiable samples was compared for each analyte and correlation analyses were performed to relate the data. For MSD S-plex, parallelism and antibody pair knockdown experiments gauged specificity of the kit.

Results: MSD S-plex was the most sensitive multiplex platform followed by Olink Target 48, Quanterix SP-X, and MSD V-plex. Concentrations across platforms differed greatly for some cytokines, but all platforms showed strong correlation. Results for MSD S-plex were confirmed by parallelism and knockdown.

Conclusion: MSD S-plex should be a priority platform for ultra-sensitive assay. Olink Target 48 offers an enticing combination of sensitivity and multiplex capability that warrants consideration when many cytokines require quantitation. MSD V-plex, MSD S-plex and Olink quantitative assays offer high utility across drug development programs, but fit-for-purpose performance should be assessed on a per-analyte basis.

Following up on our most recent discussion paper focussing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated Bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e., the what) and another on the practical considerations (i.e., the how). In this paper, we discuss 'the how'. Both papers should be helpful for the bioanalytical community to better understand the challenges and provide an insight on why bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS should not be compared with the more traditional LC-MS/MS assay for small molecules or ligand binding assays for biotherapeutics.

Aims: Circulating total desmosine, representing endogenous systemic elastin degradation activity, is an emerging biomarker for mortality risk in several diseases and aging. However, the existing analytical method takes more than 23 hours to complete, limiting its potential applications. The objective of this study was to shorten the turnover time of a stable isotope dilution liquid chromatogram mass spectrometry-based desmosine assay.

Materials & methods: Plasma samples were analyzed using acid hydrolysis followed by solid-phase extraction and LC-MS. Two approaches to reduce assay time were tested: microwave-assisted acid hydrolysis and direct injection following solid-phase extraction.

Results: The combination of acid hydrolysis at 180°C for 8 minutes and a low-volume elution design for solid-phase extraction reduced the overall assay time to ~ 30 minutes. The assay was validated with intra-day precision and accuracy ranging from 4% to 14%, and -7% to 9%, respectively, while inter-day precision and accuracy were 0% to 9% and 1% to 3%, respectively. The assay was tested in a cohort of patients with acute aortic dissection and control subjects, where desmosine concentrations were approximately three-fold higher in patients.

Conclusions: These results demonstrated that rapid desmosine analysis can be achieved with the use of both microwave-assisted hydrolysis and streamlined solid-phase extraction.