Bioanalysis最新文献

Antibody-drug conjugates (ADCs) represent a rapidly advancing class of biotherapeutics for oncology and immunological indications. Comprehensive pharmacokinetic (PK) characterization is critical for assessing ADCs efficacy, safety, and overall therapeutic performance. Ligand binding assays (LBAs) are widely employed in both academic and industrial settings for the quantitative and semi-quantitative analysis of biologics. These assays rely on specific molecular interactions - commonly between antigens and antibodies or ligands and receptors - and offer high sensitivity, robustness, and cost-efficiency. In ADC bioanalysis, LBAs are utilized to quantify multiple types of analytes, including total antibody and antibody-drug conjugate. However, the development of LBA methods for ADCs is challenged by the structural heterogeneity of these molecules, analyte instability, and the need for high selectivity and sensitivity. This review summarizes the application of LBAs in ADC PK studies, outlines common methodological challenges, and discusses strategic considerations for assay development to ensure accurate and reliable bioanalytical measurements.

Microsampling, using minute amounts of biological specimen, is uniquely suited for carrying out human and animal pharmacokinetic and toxicokinetic studies. Indeed, it provides important advantages over common blood-drawing procedures (e.g. increased analyte stability, simpler and cheaper storage and shipping, self-sampling possibility, reduced consumption of materials and animals) while also maintaining similar performance and result reliability. In the microsampling space, volumetric absorptive microsampling (VAMS) is currently considered one of the most important and widespread technologies, thanks to its volume accuracy capabilities, making it natively suitable for quantitative analysis. In this review, an exhaustive treatment of pharmacokinetic and toxicokinetic studies using VAMS is presented, as well as several examples of analytical methods prospectively enabling the employment of VAMS for the same purpose.

The development of biotherapeutics, particularly multi-domain biotherapeutics (MDBs) and antibody drug conjugates (ADCs), presents unique challenges due to their complexity and increased immunogenicity risks. Bioanalytical approaches play a pivotal role in addressing these challenges. Robust bioanalytical methods are essential for effectively assessing the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenic responses of novel biologics. Advanced techniques, such as multiplexed assays using ligand binding assays (LBAs) and Liquid Chromatography-Mass Spectrometry (LC-MS) methods, are employed to handle the complexity of MDBs and ADCs effectively. Additionally, high-resolution mass spectrometry can investigate the potential biotransformation of biologics directly in in vivo studies. Furthermore, the Context of Use (CoU) is critical for defining assay purposes across nonclinical and clinical settings, ensuring their relevance and efficiency. Regulatory compliance is paramount to ensure assays meet standards for drug approval. Practical considerations include assay sensitivity, specificity, and the method's ability to investigate the structural integrity of MDBs and ADCs in in vivo studies. This paper aims to review recent publications on the application of bioanalytical techniques, specifically LBA and LC-MS, in supporting the development of pioneering biotherapeutics. Additionally, it will discuss optimal strategies for bioanalytical methods and immunogenicity assessment.

Hybridization-based LC-MS is rapidly emerging as a bioanalytical platform for oligonucleotides, particularly when both high sensitivity and high specificity are needed. When used to analyze single-stranded antisense oligonucleotide (ASO) therapeutics, the workflows are relatively well established, but the analysis of double-stranded small interfering RNA (siRNA) therapeutics presents additional challenges due to competition for binding from the sense strand. In the last two years, the authors have independently published extensively on hybridization-based LC-MS bioanalysis of siRNA therapeutics, and now we take a step back to evaluate the progress we have made and offer our thoughts on the future of this platform. We touch upon aspects of the sample preparation and analytical process that can either be improved upon, made more efficient, or expanded to maximize the information that can be gained from a single sample. Additionally, we discuss how hybridization-based LC-MS compares to other common oligonucleotide bioanalytical workflows, and its potential to become a frontline assay platform for use in supporting regulatory submissions. Overall, we are excited about the potential hybridization-based LC-MS has demonstrated as a bioanalytical platform and are eager to begin the conversation on where this workflow goes next.

Aims: Immunogenicity testing is a key component of protein drug development, with ADA bridging assays recognized as the gold standard method. These assays employ labeled therapeutic drugs for capture and detection, with the substrate playing a critical role in generating a detectable signal to differentiate the presence of anti-drug antibodies (ADAs) from nonspecific binding. This study investigates the impact of substrate choice on the assay's ability to capture individual sample variability, focusing on screening and confirmatory cut points (SCP and CCP).

Methods: We compared the colorimetric substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with the fluorescent substrate 3-(4-hydroxyphenyl)propionic acid (HPPA) in ELISA-based ADA bridging assays for two monoclonal antibodies.

Results: Switching the substrate from ABTS to HPPA improved the differentiation of individual signals from baseline noise, enabling a more robust ADA identification. Additionally, the use of HPPA resulted in higher SCP and CCP values. Further investigation revealed that technical reader noise affected a normally distributed sample set when using ABTS.

Conclusion: The substrate switch, while maintaining all other assay parameters, proved to be a convenient approach to increase SCP and CCP while improving the ability to capture individual sample variability.

With the ongoing advancements in the field of ambient ionization for mass spectrometry, systems with a high-throughput capability on the order of 1 sample/second are readily available. This has led to the adoption of mass spectrometry for a wide variety of applications including those in the drug discovery process. Mass spectrometers have traditionally relied on pre-separation technologies such as high-pressure liquid chromatography for sample clean-up and isobaric separations, but such techniques are not high-throughput compatible. Differential mobility spectrometry is a high-speed atmospheric separation device with separations orthogonal to m/z that can be coupled with the high-throughput sample introduction devices such as acoustic ejection mass spectrometer to address this gap. In this article we highlight the significance of the recent reports on this topic and provide some insights into expanding the use of this technique for new applications. We believe this is a promising new development and will help propel the high-throughput mass spectrometry beyond isobaric interferences.

Aims: To establish a rapid, sensitive HPLC-MS/MS method for quantifying isavuconazonium in human plasma and characterize its pharmacokinetics in healthy Chinese subjects under fasting and postprandial conditions.

Materials & methods: Plasma samples were processed via acetonitrile protein precipitation. Separation was performed on an LC-20ADXR Plus C18 column with gradient elution (0.01% formic acid/acetonitrile). Detection used a Triple Quad 4500 mass spectrometer with MRM; isavuconazole-d4 was the internal standard. The method was validated, then applied to a crossover study in 32 healthy subjects (fasting vs. postprandial).

Results: The method showed good linearity (4-4000 ng/mL, R² ≥0.9801) with LLOQ 4 ng/mL. Stability was confirmed under various conditions (e.g. 53 days at -20°C, 66 days at -80°C). Pharmacokinetic results revealed food delayed Tmax (2.5 vs. 5.0 h) and reduced Cmax (1929.68 vs. 1300.17 ng/mL) but did not affect AUC_0-t.

Conclusions: The validated HPLC-MS/MS method is rapid and reliable for therapeutic drug monitoring. Food affects absorption but not total exposure, guiding clinical dosing in Chinese populations.

Background: Drug bridging immunoassays are widely employed as the standard approach for detecting anti-drug antibodies (ADAs) in the development of new biological entities. A major challenge in these assays is mitigating target interference, particularly when the soluble target exists in dimeric forms, which can result in false positive signals and compromise assay specificity.

Research design and methods: We developed sensitive and robust ADA assays capable of overcoming target interference to detect antibodies against BI X in both cynomolgus monkey (cyno) plasma and human serum matrices. This was achieved through the implementation of simple sample treatment techniques, specifically, acidification using a panel of different acids, to disrupt dimeric target interactions and minimize the interference.

Results: Optimization of the acid dissociation and subsequent neutralization steps significantly reduced target interference in both cyno and human matrices. These improvements were achieved without the need for additional assay development or complex depletion strategies.

Conclusions: Compared to previously reported methods for mitigating target interference, the acid panel treatment approach is simpler, more time-efficient, and cost-effective. This user-friendly strategy can be readily applied to eliminate soluble dimeric targets during ADA method development, particularly in cases where alternative methodologies are not feasible or applicable.

Introduction: Oral metronomic chemotherapy employs a low-dose combination of chemotherapeutics administered regularly to minimize toxicity while enhancing anticancer efficacy. The clinical utility of Doxorubicin (DOX) is limited due to severe cardiotoxicity. Interestingly, Piperine (PIP) has been explored to mitigate DOX-induced toxicity while enhancing its therapeutic efficacy. Solid lipid nanoparticles (SLNs) offer an efficient drug delivery approach to improve oral bioavailability and controlled release of DOX and PIP.

Areas covered: LC-MS/MS method was developed and validated per US-FDA bioanalytical guidelines to quantify DOX and PIP in plasma simultaneously. SLNs were developed and optimized using design expert software and exhibited particle size of 151.56 ± 0.32 nm, polydispersity index (PDI) of 0.172 ± 0.02, and surface charge of -22.83 ± 0.66 mV. Pharmacokinetic evaluation in female Sprague-Dawley rats showed enhanced AUC₀-∞ for DOX (31911.78 ± 226.92 ng/mL) and PIP (7377.66 ± 655.78 ng/mL), indicating improved systemic exposure.

Expert opinion: The findings highlight the potential of SLN-based co-delivery of DOX and PIP for oral metronomic chemotherapy. The validated LC-MS/MS method ensures precise pharmacokinetic assessment, which is crucial for future clinical translation. This study provides a promising strategy for enhancing oral chemotherapy efficacy.

Aim: To confirm the observation of IgM interference in the anti-adeno-associated virus (AAV) IgG immune complex (IC) assay format and to verify that IgM-specific digestion can improve anti-AAV IgG detection in IC assays.

Methods: Treatment-emergent anti-AAV2 and anti-AAV9 IgG signals were measured in IC assays with and without IgM-specific digestion. Anti-AAV2 and anti-AAV9 IgM signals were measured in parallel.

Results: IgM digestion increased anti-AAV2 and anti-AAV9 IgG signals when anti-AAV2 or anti-AAV9 IgM were present in the matrix.

Conclusions: Co-existing anti-AAV IgM cause interference in the anti-AAV IC assay format. Selective IgM digestion improves the detection of anti-AAV IgG in the IC assay.