Tierney B, Tuttle PG, Karahanoglu FI
… +12 more, Reyes CR, Chen Y, Blatnik M, Vigil S, Ziegenfelder M, Santamaria M, Chou DE, Williams B, Gao A, Peterson B, Adhin B, Cai X
Bioanalysis
· 2025 Jun · PMID 40495533
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AIM: This study assessed the agreement and bias of three dried blood patient-centric microsampling (PCS) devices (paper DBS, Mitra, and Tasso-M20) for the quantitation of branched chain amino acids (BCAAs) and ketoacids...AIM: This study assessed the agreement and bias of three dried blood patient-centric microsampling (PCS) devices (paper DBS, Mitra, and Tasso-M20) for the quantitation of branched chain amino acids (BCAAs) and ketoacids (BCKAs) compared to venous plasma samples. MATERIALS & METHODS: Concentrations of BCAAs and BCKAs were measured in samples from generally healthy participants with a validated assay for venous plasma adapted for use with dried blood from the PCS devices. Participants were also asked to respond to a device usability questionnaire about their experience with the devices. RESULTS: The correlations between BCAA/BCKA concentration values in PCS devices vs venipuncture samples were strong to excellent for all six analytes, across all three PCS devices. The surrogate analyte approach facilitated method transfer from plasma to dried blood microsampling devices. Overall, participants reported that they enjoyed using the devices compared to traditional venipuncture and would be willing to use them again, on their own at-home. CONCLUSION: These study results demonstrate the feasibility of quantitating BCAAs and BCKAs reliably with PCS devices, with high acceptability from study participants.
Bioanalysis
· 2025 May · PMID 40492479
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AIMS: Design of experiments (DOE) is a versatile and efficient approach to tackle complex scientific problems. We aimed to assess its feasibility in the optimization of the multistep, involved bottom-up sample preparatio...AIMS: Design of experiments (DOE) is a versatile and efficient approach to tackle complex scientific problems. We aimed to assess its feasibility in the optimization of the multistep, involved bottom-up sample preparation for the UPLC-MS/MS analysis of proteins. MATERIALS AND METHODS: The model analyte was a human IgG1 monoclonal antibody which was spiked into rat plasma and processed further by reduction, alkylation, and digestion for the subsequent UPLC-MS/MS analysis. The Modde Go software was used for the generation of experimental designs and for processing, analyzing and the interpretation of the data. RESULTS: DOE screening revealed that urea made the biggest improvement on the surrogate peptide responses, while guanidine significantly suppressed them. DOE optimization resulted in a 2-, 10-, 10- and 50-fold response increase for the respective DTLM, FNWY, TPEV and VVSV surrogate peptide even after a short, <3-h sample preparation, when compared to a legacy method that required 2-day preparation. CONCLUSIONS: The DOE approach was applied successfully for the comprehensive optimization of eight denaturation, reduction and digestion parameters. DOE was found to be an efficient tool for protein sample preparation optimization, and the predictive power of the DOE models was successfully demonstrated.
Bioanalysis
· 2025 May · PMID 40471672
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Folates are group of water-soluble B-vitamins indispensable to one carbon metabolism as acceptor and donor of methyl group during purine and pyrimidine biosynthesis, DNA and histone methylations, and in mitochondrial pro...Folates are group of water-soluble B-vitamins indispensable to one carbon metabolism as acceptor and donor of methyl group during purine and pyrimidine biosynthesis, DNA and histone methylations, and in mitochondrial protein translation. The deficiencies associated with risk of neural tube defect, cancer, cardiac and psychiatric disorders. Thus, detecting and quantifying folate species accurately become a crucial step in food omics, disease metabolomics, proteomics, genomics, toxicology, and pharmacokinetics, and regulatory sciences. However, the detection and quantitative determination of folate species yet subjected to analytical challenges due to physio-chemical instability, structural similarity, ultra-trace availability. Advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) method enabled the detection and quantification of folate species in short span of time using low sample volume. However, risk of inter conversion, degradation or loss during sample preparation, coupled with folate isomers and isobars challenged the selectivity, specificity and sensitivity for quantification by LC-MS/MS at trace level. Systematic literature search was conducted through major indexing databases such as Pub med, Embase, and Google Scholar to include the most relevant articles published 2010-2025 in preparing the review highlighting the challenges of folate species analysis in food and biological matrices from sample preparation to mass spectrometry detection with a future perspective on innovative optimization methods.
Niwa M, Noda K, Kobayashi Y
… +2 more, Kageyama I, Kodama K
Bioanalysis
· 2025 May · PMID 40462640
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PURPOSE: Using two diseases, Alzheimer's disease and muscular dystrophy as examples, in which many resources have been invested, trends in bioanalytical techniques for detection of peptide or protein biomarkers using mas...PURPOSE: Using two diseases, Alzheimer's disease and muscular dystrophy as examples, in which many resources have been invested, trends in bioanalytical techniques for detection of peptide or protein biomarkers using mass spectrometry are reviewed using Web of Science database. Amyloid beta peptides have been used to detect Alzheimer's disease, and peptides obtained by tryptic digestion of dystrophin have been used to detect muscular dystrophy. Based on the recent interest in peripheral blood analysis, literature review was focused on biofluid analysis. RESULTS: Both electrospray or matrix-assisted laser desorption ionization mass spectrometry were employed for amyloid beta peptides, and high-resolution or ion mobility techniques had been introduced to enhance selectivity. Immunoprecipitation techniques were often used for pretreatment; however, emergence of combined use of different solid-phase extraction mode was observed. Regarding tryptic peptides obtained from dystrophin, there are few reported applications in biofluids, and typically electrospray ionization-selected reaction monitoring with pretreatment using solid-phase extraction were employed. IMPLICATIONS: Validity of blood biomarkers propelled development of bioanalytical methods by attracting research interest. The analytical methodologies was built on proven methods in the field of peptide analysis, and current interests were in techniques increasing selectivity and sensitivity, such as differential mobility spectrometry or parallel reaction monitoring.
Chen H, Liu K, Zhang J
… +4 more, Zhao X, Wang Y, Lu X, Qin F
Bioanalysis
· 2025 May · PMID 40420470
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AIM: A rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for epalrestat detection in human plasma. MATERIALS AND METHODS: A triple...AIM: A rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for epalrestat detection in human plasma. MATERIALS AND METHODS: A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used to quantify epalrestat and the internal standard epalrestat-d in the negative ion mode using multiple reaction monitoring (MRM). After acetonitrile-mediated protein precipitation, chromatographic separation was achieved using a reversed-phase C column (ACQUITY UPLC BEH, 2.1 × 50 mm, 1.7 μm; Waters Corp) with acetonitrile and 2 mM ammonium acetate in water as the mobile phase through gradient elution. RESULTS AND CONCLUSION: The retention time of epalrestat was 0.92 min and the entire run time was only 2.00 min. The calibration curve was linear in the range 10.0-8.00 × 10 ng/mL ( ≥ 0.99). The within-run and between-run relative standard deviations (RSDs) were < 9.3%. The within-run and between-run relative errors (REs) ranged -8.4-4.2%. No significant matrix effects were observed and the recovery rate was high. The method was fully validated, including reinjection reproducibility in human plasma, and was successfully applied in a pharmacokinetic study, in which 100% incurred sample reanalysis met the criteria.
Borges GR, Dos Santos BP, de Gouveia GC
… +4 more, Dalanhol CS, Scherer JN, Eller S, de Oliveira TF
Bioanalysis
· 2025 May · PMID 40401753
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BACKGROUND: Oral fluid has proven to be an excellent matrix for drug testing. Several analytical techniques are available, including the use of dried spots, which have gained increasing recognition in recent years. In th...BACKGROUND: Oral fluid has proven to be an excellent matrix for drug testing. Several analytical techniques are available, including the use of dried spots, which have gained increasing recognition in recent years. In this study, a new method was developed and validated using dried oral fluid spot (DOFS) assisted by a 24-well plate for the simultaneous determination of thirteen drugs of abuse and metabolites in oral fluid by LC - MS/MS. METHODS: Samples were collected by expectoration and diluted with water. A 100 μL aliquot of sample was applied onto the spot, dried for 60 min, and extracted with 250 μL of acetonitrile:methanol (3:1). The method was validated according to the ANSI/ASB Standard 036 guideline, and 86 oral fluid samples were analyzed. RESULTS: The limits of quantification ranged from 1 to 5 ng/mL, and all calibration curves were linear. Precisions ranged from 2.0 to 13.0% and Bias fluctuated from -19.7 to 9.6%. Ionization suppression or enhancement was noted at -16.2 to 16.6%. Among the analyzed samples, 86% tested positive for at least one substance. CONCLUSIONS: The findings confirm that the proposed method was successfully validated, providing a new bioanalytical approach for the detection of drugs of abuse in oral fluid.
Abrigo N, Selaya SD, Mohammad A
… +12 more, Shakleya D, Brown DG, Zhang J, Pratt V, Green JE, Degunia A, Bennett M, Bloom K, Rogers L, Khan AQ, Heuckeroth RO, Faustino PJ
Bioanalysis
· 2025 May · PMID 40401362
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BACKGROUND: While Polyethylene glycol 3350 (PEG 3350) is approved by the USFDA for short term use by adults, it is commonly recommended for use in constipated children. Multiple reports of adverse events in children taki...BACKGROUND: While Polyethylene glycol 3350 (PEG 3350) is approved by the USFDA for short term use by adults, it is commonly recommended for use in constipated children. Multiple reports of adverse events in children taking PEG 3350 raised safety concerns suggesting that low molecular weight species of PEG 3350 might be absorbed from the gut and cause side effects such as ethylene glycol (EG), diethylene glycol (DEG), and triethylene glycol (TEG). RESEARCH DESIGN AND METHODS: This article documents the development, validation, and application of analytical methods using GC-MS and GC-MS/MS for the quantitation of EG, DEG, and TEG in human plasma, serum, and urine. RESULTS: The analytical range for EG, DEG, and TEG was 2-20 µg/mL. The sample preparation process involves derivatization using N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane in each biological matrices. Deuterated internal standards for each of the analytes were included to provide accurate quantitation of the glycol analytes. CONCLUSIONS: The validated methods were applied to analyze samples a pilot study of children taking PEG 3350. DEG and TEG were detected at levels below the limit of quantitation. In summary, a platform of analytical methods was developed to evaluate glycol analogs in urine, serum, and plasma clinical samples.
Luo K, Liu D, Zhang S
… +4 more, Zhang X, Cheng Q, Lin J, Yu Y
Bioanalysis
· 2025 May · PMID 40371913
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AIMS: This study aimed to establish a robust LC-MS/MS method for quantifying trazodone in human plasma and investigate its pharmacokinetic profile in healthy Chinese subjects under fasting and fed conditions. MATERIALS &...AIMS: This study aimed to establish a robust LC-MS/MS method for quantifying trazodone in human plasma and investigate its pharmacokinetic profile in healthy Chinese subjects under fasting and fed conditions. MATERIALS & METHODS: A validated LC-MS/MS method using protein precipitation was applied to analyze trazodone in 68 healthy subjects (34 fasting/34 fed), with full method validation performed. RESULTS: This method exhibited high selectivity, precision, and accuracy. The results showed good linearity in the range of 5-3000 ng/mL and matrix effect results showed no matrix interference. The pharmacokinetic data revealed notable differences between the fasting and postprandial states. CONCLUSIONS: The validated LC-MS/MS method proved reliable for trazodone quantification. The observed food effects on absorption suggest clinical dosing should consider administration conditions to ensure optimal therapeutic outcomes.
Yan X, Wang S, Li Y
… +7 more, Cui L, Zhu Z, Liu H, Wang L, Zhu Y, Zhao F, Yang P
Bioanalysis
· 2025 Apr · PMID 40371815
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AIMS: Omega-3 (-3) and omega-6 (-6) polyunsaturated fatty acids (PUFAs) are indispensable for human health. Notably, imbalanced levels of maternal PUFAs during pregnancy can exert lasting impacts on the developing fetus....AIMS: Omega-3 (-3) and omega-6 (-6) polyunsaturated fatty acids (PUFAs) are indispensable for human health. Notably, imbalanced levels of maternal PUFAs during pregnancy can exert lasting impacts on the developing fetus. There is an urgent need for a robust and rapid method that can simultaneously quantify multiple -3 and -6 PUFAs in plasma and adapt to large-scale clinical screening. METHODS: Sample preparative process consisted of non-derivatized alkali hydrolyzing and liquid-liquid extraction followed by reconstituting after being blown dry with nitrogen, and the total chromatographic run time was only 5.6 min. RESULTS: The LC-MS/MS method was validated according to current national and international guidelines. Acceptable reproducibility and accuracy were shown based on the intra-day precision was 1.79%-7.02%, the inter-day precision was 0.01%-5.63%, and the accuracy was 87.42%-102.78%. The correlation coefficients of linearity were >0.9930. All measured plasma individual PUFAs levels in pregnant women significantly increased, apart from eicosapentaenoic acid (EPA, C20:5n3) and -6/n-3 PUFA ratio levels decreased were observed. The analysis performance is satisfactory and suitable for clinical routine detection. CONCLUSION: This method could be a valuable tool for 10 kinds of -3 and -6 PUFAs nutritional assessment, supplementary guidance, and intervention during pregnancy.
Bioanalysis
· 2025 May · PMID 40366769
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A highly sensitive and selective LC-MS/MS assay was developed and validated for the simultaneous quantification of Chlordiazepoxide and Clidinium in human plasma for the first time, employing solid-phase extraction. Chro...A highly sensitive and selective LC-MS/MS assay was developed and validated for the simultaneous quantification of Chlordiazepoxide and Clidinium in human plasma for the first time, employing solid-phase extraction. Chromatographic separation of the analytes and their deuterated internal standards was performed on a reversed-phase Kinetex XB-C18 (150 × 4.6 mm, 5 μm) column with a gradient mobile phase. Mass spectrometric detection was achieved using electrospray ionization in positive ion mode, employing the ion transitions: m/z 300.0 → 227.1 for Chlordiazepoxide, m/z 352.1 → 142.1 for Clidinium, m/z 305.1 → 232.1 for Chlordiazepoxide D5, and m/z 357.2 → 142.2 for Clidinium D5. The assay demonstrated a linear calibration range of 504.0-500,198.3 pg/mL for Chlordiazepoxide and 5.0-3,004.7 pg/mL for Clidinium, ensuring precise pharmacokinetic evaluation. The method was validated with a lower limit of quantification (LLOQ) of 504.0 pg/mL for Chlordiazepoxide and 5.0 pg/mL for Clidinium, precision within 15% RSD, and accuracy within 85-115% of the nominal values. No matrix interference from haemolysed or lipemic plasma was observed, and recovery exceeded 90%. This study presents a novel LC-MS/MS method with significant improvements in sensitivity and specificity, facilitating its direct application in pharmacokinetic and bioequivalence studies.
Ginting ENB, Lembang GV, Abdullah DAP
… +3 more, Nurrakhma SSR, Ridwan MRI, Permana AD
Bioanalysis
· 2025 May · PMID 40366746
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BACKGROUND: Rifampicin (RIF) is the main treatment for tuberculous meningitis (TBM), but its limited penetration across the blood-brain barrier significantly reduces its therapeutic efficacy. To address this limitation,...BACKGROUND: Rifampicin (RIF) is the main treatment for tuberculous meningitis (TBM), but its limited penetration across the blood-brain barrier significantly reduces its therapeutic efficacy. To address this limitation, smart-pH nanoparticles were designed to release RIF in inflamed area and incorperated into a thermosensitive in situ gel for enhanced drug delivery via the olfactory nerve. METHODS: UV-Vis spectrophotometry and colorimetric methods to quantify RIF in various media and biological matrices, including phosphate-buffered saline (PBS) at pH 7.4 and 5.0, plasma, and brain tissue. The main validation parameters assessed were selectivity, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, extraction recovery, and dilution integrity. RESULT: Method validation followed International Council for Harmonization (ICH) guidelines, demonstrating excellent linearity (r = 0.999) and lower limit detection (LOD) of approximately 0,25-0.49 μg/mL were achieved in all media. The method demonstrated high accuracy (%RE -11.62% to 14.88%) and precision (%RSD 2.06% to 13.29%), meeting regulatory requirements. CONCLUSION: This validated approach enables reliable RIF quantification in biological samples, supporting its application in in vitro release studies, ex vivo permeability studies, and in vivo pharmacokinetic evaluations. These findings contribute to the advancement of targeted drug delivery systems for TBM treatment.
Lepers C, Bellanger A, Petrof O
… +3 more, Verniquet M, Le Bouter M, Taverne C
Bioanalysis
· 2025 May · PMID 40357562
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Assessing the performance of an immunogenicity assay before using it to support vaccine clinical trials is mandatory. Further validation of assay performance is even requested to support Phase III studies. While assay va...Assessing the performance of an immunogenicity assay before using it to support vaccine clinical trials is mandatory. Further validation of assay performance is even requested to support Phase III studies. While assay validation requires adherence to predefined acceptance criteria, there is no universal stringency level for earlier phases of assay development. Setting scientifically sound criteria for success is about finding the right balance between unavoidable assay limitations and ensuring the assay is fit-for-purpose. In this paper, we focus on the evaluation of linearity for immunogenicity assays and its use as surrogate for accuracy evaluation in the absence of reference material. We propose a simple method for evaluating assay linearity and understanding the impact of a linearity deviation on the evaluation of the response increase in clinical trial. Assessment is reliable in absence of known concentration samples, unlike methods in industry guidance, making the proposed method more versatile. Method is illustrated by two case studies, representative of the assays used in the vaccine field. Making the link between assay linearity and clinical endpoints provides a simple and clear framework to support the definition of assay validation criteria, applicable to a variety of immunogenicity assays.
Bioanalysis
· 2025 Apr · PMID 40346877
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AIM: Monitoring immune responses to therapeutic peptides with endogenous counterparts is crucial for evaluating drug safety and efficacy. In this paper, we focused on the selection of an optimal assay format to develop a...AIM: Monitoring immune responses to therapeutic peptides with endogenous counterparts is crucial for evaluating drug safety and efficacy. In this paper, we focused on the selection of an optimal assay format to develop a sensitive, robust, and drug-tolerant immunoassay for the detection of anti-drug antibody (ADA) against a therapeutic peptide. RESULTS: We assessed distinct ADA assay formats for preclinical and clinical studies, such as direct binding with labeled protein A/G, direct binding with labeled multiple species-specific antibodies for detection, bridging and affinity capture elution (ACE) formats. The assay formats were evaluated based on multiple assay parameters including sensitivity, drug tolerance, individual matrix variability and inter-assay precision. Overall, direct binding assay with labeled protein A/G for detection, which utilized less labeled peptide drug and achieved desired sensitivity and drug tolerance, is appropriate for preclinical studies. Bridging assay is more suitable format to support clinical studies as bridging assay has less assay variability than ACE assay. CONCLUSION: This study highlighted advantages and limitations of each ADA assay format for peptide drugs and evaluated the performance of different assay formats in the assay development process to aid in the selection of the best fit-for-purpose assay formats for preclinical and clinical phases.
Javid S, Ramya K, Gulzar Ahmed M
… +7 more, Zahiya N, Thansheefa N, Anas AK, Mahfeela F, Reeha, Sha F, Sultana R
Bioanalysis
· 2025 Apr · PMID 40256889
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Hypertension is a multifaceted cardiovascular disease, a significant risk factor for stroke, heart attack, heart failure, and renal damage. An essential phase in the drug development process is the exploration of effecti...Hypertension is a multifaceted cardiovascular disease, a significant risk factor for stroke, heart attack, heart failure, and renal damage. An essential phase in the drug development process is the exploration of effective bioanalytical approaches to investigate drug metabolism and pharmacokinetics precisely. The use of LC-MS has increased significantly over the last 10 years; numerous researchers have made contributions to the field and enhanced the technical capabilities of workflows based on LC-MS. This review provides a critical analysis of the method development and validation of bioanalytical methods using Liquid Chromatography-Mass Spectrometry (LC-MS) of a few antihypertensive drugs, focusing on extraction techniques and validation parameters. Furthermore, a fleeting look at the GLP, regulatory guidelines, machine learning and artificial intelligence in bioanalysis. Despite these advancements, the document identifies gaps in current regulatory guidelines and advocates areas for further research, predominantly concerning matrix effects and the impact of co-medications. The integration of AI tools in LC-MS has shown the potential to revolutionize bioanalytical methods, yet there is still an imperative for global harmonization. We assume that this review will offer a foundation for the research of new strategies and assist in the identification of the optimum relevant methodology parameters for known and emerging antihypertensive drugs.
Yuan M, Liu A, Xu B
… +64 more, Sales K, Karnik S, Voelker T, Salha D, Zimmer J, O'Dell M, Gorityala S, Hays A, Lester T, Reynolds G, Tary-Lehmann M, Yu M, Roberge M, Malone K, Patel V, Love I, Lin J, Diaz M, Xu T, Garofolo W, McGregor J, Leskovar A, Kernstock R, Pellerin M, Brown M, Spytko A, Lowes S, Ambrose D, Dufield D, Kane C, Veeramachaneni R, Luna M, Warrino D, Dwivedi V, Xu A, Hyer E, Iles T, Majumdar R, Sikkema D, Thomas E, Carlsson A, Dakappagari N, Riccitelli N, Marco CD, Bouhajib M, Iordachescu A, Sanghvi M, Barton H, Lavelle A, Dompkowski E, Rundlett S, Matys K, Sangster T, Turksma A, Gu W, Liu J, Hoffpauir B, Rocha A, Pirro J, Bergeron J, O'Brien K, Fang X, Dong K, Yamashita J
Bioanalysis
· 2025 Apr · PMID 40248960
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Biomarker analysis enables a deep understanding of physiological and biological processes and offers insights into pathological disease states and conditions. When measured in tissues, the spatial distribution of biomark...Biomarker analysis enables a deep understanding of physiological and biological processes and offers insights into pathological disease states and conditions. When measured in tissues, the spatial distribution of biomarkers may be evaluated. To meet regulatory and sponsor requirements, guidance on the approach to validation and the parameters to be evaluated is essential. The main goals of this GCC white paper are to disseminate the survey results discussed during the 16& 17GCC Closed Forums (2023 & 2024) and to provide recommendations from the GCC members on technical and regulatory considerations for the bioanalysis of biomarkers in tissues.
Nishio R, Nakanaga K, Yoshinaga R
… +2 more, Kurihara A, Okano F
Bioanalysis
· 2025 Apr · PMID 40243353
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AIM & METHODS: TRK-950, a humanized IgG1 monoclonal antibody against CAPRIN-1, which is reported to be expressed on the cell surface of various forms of cancer, is currently being developed for multiple types of solid ca...AIM & METHODS: TRK-950, a humanized IgG1 monoclonal antibody against CAPRIN-1, which is reported to be expressed on the cell surface of various forms of cancer, is currently being developed for multiple types of solid cancer. We developed an antidrug antibody (ADA) assay that includes two pretreatment processes for use in clinical trials, namely, Melon™ gel treatment and solid-phase extraction with acid dissociation (SPEAD), to reduce baseline variability between individual serum samples and improve drug tolerance. RESULTS: Comparing the assay with or without the pretreatment process using commercially available human serum samples, the mean response calculated from 18 individual human samples decreased from 327 to 270, and the coefficient of variation decreased from 41% to 16%. The new assay with the two-step pretreatment met the criteria set by the regulatory agency throughout the validation study. We also confirmed the recovery rates of IgG, IgM, and ADA - drug immune complexes after treatment with Melon™ gel. Clinical trials (NCT05423262) employing this improved analytical method have been conducted, and the results confirm the low immunogenicity of TRK-950. CONCLUSION: The combination of pretreatment with Melon™ gel and SPEAD is applicable for clinical ADA assays.
Ieki K, Fukuda S, Miyawaki S
… +1 more, Hirowatari K
Bioanalysis
· 2025 Apr · PMID 40205765
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Antibody-drug conjugates (ADCs) are emerging as powerful tools in cancer therapy. Evaluating their drug disposition requires the development and validation of analytical methods to obtain accurate quantitative results, w...Antibody-drug conjugates (ADCs) are emerging as powerful tools in cancer therapy. Evaluating their drug disposition requires the development and validation of analytical methods to obtain accurate quantitative results, which depend on understanding the ADC structural properties and selecting appropriate analytical platforms. Liquid chromatography-mass spectrometry (LC-MS) is a key technology for ADC bioanalysis, enabling the quantification of payloads, linkers, total antibodies, ADCs, and drug-to-antibody ratio (DAR). This review highlights the strategies and challenges in developing analytical methods for quantifying ADC components in biological samples using LC-MS with a focus on their constituent units. In addition, it addresses the validation requirements of these quantitative analytical methods during drug development.
Cowan KJ, Bloem K, Coddens A
… +13 more, Creed L, Higgins H, Novoa JJ, Jones C, Katterle Y, Klinge M, Leatherdale B, Jaitner B, van Bommel P, van der Lee S, Golob M, Nelson R, Timmerman P
Bioanalysis
· 2025 May · PMID 40200843
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The European Bioanalysis Forum, in discussions with cross-industry experts from pharmaceutical, biotech, and contract research organizations, is continuing the challenge of the traditional 3-Tier immunogenicity testing s...The European Bioanalysis Forum, in discussions with cross-industry experts from pharmaceutical, biotech, and contract research organizations, is continuing the challenge of the traditional 3-Tier immunogenicity testing strategy, proposing a simpler, context-driven 1-Tier approach, a recent paradigm shift that emphasizes clinical relevance and the impact of anti-drug antibodies over mere incidence. In a workshop at the 17 annual Open Symposium, the meeting highlighted using signal-to-noise ratios to assess low-level ADA responses and addressed challenges such as nonspecific binding and assay reliability. Participants debated the need for continued confirmatory testing, and that it should be performed on a case-by-case basis. The shift aims to improve clinical testing strategies while reducing complexity, promoting data-driven decisions. Case studies, particularly from high-risk molecules, will guide implementation. Overall, there's strong support for the 1-Tier approach, but its success depends on robust data, industry collaboration, and regulatory approval.