Bioanalysis最新文献

Background: Cell and Gene Therapy (CGT) utilizes a nucleic acid core, which makes quantitative PCR (qPCR) a critical tool for establishing pharmacokinetics profiles supporting preclinical and clinical studies of CGT programs. Recently, digital PCR (dPCR) has gained traction in the CGT space.

Aim: Here we report a comparison of assay performance between qPCR and a nanoplate-based digital platform in a mouse biodistribution study.

Methods: This study adapted a validated duplex qPCR method for quantifying gene therapy genome copy numbers to a nanoplate-based digital PCR method. The assay performance and bioanalysis results with both methods were compared.

Results: The qPCR assay exhibited a broader dynamic range (up to 1e8 copies per reaction) while dPCR covered up to 5e6 copies per reaction with dilutional linearity. Both methods showed comparable Limit of Detection and assay sensitivity. The dPCR demonstrated a superior recovery of spiked targets in matrices, including off-target tissues, with only 1/10 of the input matrix.

Conclusions: Both platforms pose strengths and limitations. The dPCR offers a better option for biodistribution studies for its overall superior recovery in tissues tested. With a wider dilutional linearity, the dPCR may serve as a preferable option for preclinical biodistribution studies.

Aim: The therapeutic effectiveness of a drug could be significantly enhanced by prolonging its retention time. Polyethylene glycosylation (PEGylation) technology is a method to increase the half-life of protein therapeutics. PEGylated anti-Factor D Fab (PEG-aFD) was developed as a potential therapeutic, targeting factor D to reduce the frequency of intravitreal injections in patients with geographic atrophy. To support a GLP Toxicology study, there was a need to develop a PEG assay to measure total PEG concentration.

Result: PEG-aFD consists of Fabs conjugated to a multi-arm PEG molecule, which posed challenges for PEG assay development. A sensitive fit-for-purpose, semi-homogeneous competitive ELISA (Enzyme-Linked Immunosorbent Assay) using a biotinylated version of the therapeutic as the competing molecule was successfully developed and validated with LLOQ (lower limit of quantification) at 200 ng/mL. The assay was used to measure total PEG concentrations in cynomolgus monkey serum samples from the study. The results revealed measurable PEG levels (ranging from 0.468 μg/mL to 188 µg/mL) in all samples across all dosage levels, but no PEG-related toxicity was observed in all the animals.

Conclusion: A competitive ELISA was successfully developed and validated to measure total PEG concentrations in cynomolgus monkey serum samples to support a GLP Toxicology study.

Aim: The purpose of this work was to optimize the sensitivity and the drug tolerance of two cell-based assays for the detection of neutralizing antibodies (nAbs) to semaglutide (a GLP-1 analogue) and to endogenous GLP-1.

Methodology: The two assays were developed and validated in three distinct iterations. Enhancements in sensitivity and drug tolerance were achieved through platform optimization, sample pre-treatment and the alteration of control antibodies.

Results: The sensitivity and drug tolerance improved gradually with the different versions of the assays. For detection of nAbs to semaglutide, sensitivity was improved from 3,400 ng to 98 ng/ml antibody, and drug tolerance was improved from 2.5 nM semaglutide when detecting 3,400 ng/ml antibodies to 4.8-5.6 nM semaglutide when detecting 1,000 ng/ml antibody. For the endogenous GLP-1 assay, sensitivity was improved from 6,900 ng/ml to 46 ng/ml antibody, and drug tolerance improved from 1.0 nM semaglutide when detecting antibody concentration of 8,800 ng/ml to 2.5 nM semaglutide when detecting 1,000 ng/ml antibody.

Conclusion: Key factors for enhancing the sensitivity and drug tolerance of the assays included the concentration of the drug standard for receptor activation, pre-treatment of the samples, and better understanding of the binding properties of the control antibody.

Infectious diseases have a wide range of significant characteristics with complex clinical manifestations. The incidence rate and mortality rate remain high, seriously endangering human health. Therefore, rapid identification and sensitive diagnosis of pathogens are crucial for patient recovery. In order to overcome the limitations of traditional cultivation methods, such as long detection time and limited microbial species, various new detection technologies have rapidly developed in recent years. This not only greatly shortens the detection time, but also effectively improves the accuracy and sensitivity of pathogen detection. This article reviews the rapid detection methods for pathogens that are currently in the research process or early application stage in recent years, including detection technologies in molecular biology, immunology, biosensors, and microfluidics. It focuses on summarizing the sample pretreatment method, detection principles, mechanisms, and advantages of various technologies, and analyzes the current application status of various technologies in pathogen detection in clinical biological samples, in order to provide technical references for rapid detection of different clinical biological samples.

Background: CB-1 is a novel compound evaluated in Sprague Dawley rats to assess its acute oral safety and pharmacokinetic disposition.

Objectives: To determine the short-term oral toxicity of CB-1 and characterize its pharmacokinetic parameters.

Methods: Acute oral toxicity was tested at doses up to 100 mg/kg. For pharmacokinetics, six rats received a single 100 mg/kg oral dose. Plasma samples were analyzed using a polar C18 column and quantified by LC - MS/MS monitoring the 490.063→262.00 m/z transition.

Results: No mortality occurred at 100 mg/kg, indicating acceptable acute safety. CB-1 reached a peak plasma concentration (C_max) of 4324.24 ng/mL at 1 h (T_max1). Pharmacokinetic analysis revealed an apparent clearance (Cl/F) of 3.6 L/h, elimination half-life (t_1/2) of 7.86 h, and volume of distribution (Vd/F) of 40 L. A secondary plasma concentration peak at 8 h (T_max2) suggested enterohepatic recirculation.

Conclusions: CB-1 demonstrated short-term oral safety with rapid absorption, relatively low clearance, and prolonged systemic exposure, likely influenced by enterohepatic recirculation.

Aims: Accurate and precise measurement of dried blood spot (DBS) phenylalanine (Phe) is vital for managing phenylketonuria (PKU). Standard DBS collection devices use grade-226 filter-paper, while the CapitainerB quantitative device utilizes grade-222 filter-paper. Although grade-226 filter-paper performance is well characterized, data on grade-222 filter-paper are sparse. This study aimed to investigate the analytical properties of grade-222 and grade-226 filter-papers.

Materials and methods: We compared grade-222 and grade-226 filter-papers for Phe measurement accuracy and imprecision in DBS generated using both filter-papers. Scanning electron microscopy (SEM) and slit lamp imaging were used to assess the physical properties of the filter-papers.

Results: Using an aqueous calibrator as reference, grade-222 exhibited a mean bias of -1.1%, the mean bias for grade-226 was -7.3%. Intra-assay imprecision was 2.3% for grade-222, versus 4.2% for grade-226. SEM revealed that fibers in grade-226 filter-paper are bonded by an amorphous material, which is absent in grade-222 filter-paper. Total error analysis indicated grade-222 filter-paper reduced uncertainty of Phe measurement compared to grade-226 filter-paper.

Conclusions: Grade-222 filter-paper was proven to have superior analytical performance for Phe quantification, providing improved differentiation between safe and harmful Phe concentrations and offering more reliable PKU monitoring compared to traditional grade-226 filter-paper.

Aim: This study aimed to establish a method for the simultaneous quantification of 7 bile acid components in mouse feces and to apply this method to investigate the variations in bile acid profiles among control, DSS, and mice treated with Alpiniae Oxyphyllae Fructus extract (AOE). The findings may provide insights into the potential regulatory effects of AOE on bile acid metabolism in ulcerative colitis (UC).

Methods: A murine model of UC was established by dextran sulfate sodium (DSS) induction. Following treatment with AOE, fecal samples were collected from each experimental group. Mouse fecal samples were extracted with methanol, and bile acid concentrations were determined using UPLC-MS/MS with chenodeoxycholic acid-d4 as the internal standard. Separation was performed on a Kinetex® 2.6 μm C18 column (50 mm × 2.1 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile. An electrospray ion source was employed to scan in multiple reaction monitoring mode in negative ion mode.

Results and conclusion: The method established by our research institute is simple to operate, with high accuracy, sensitivity, and strong specificity, enabling rapid quantification of seven bile acid components in mouse feces. Fecal analysis revealed that bile acid alterations are associated with UC progression and suggesting AOE's therapeutic potential.

Background: Event-free survival (EFS) in patients with B-cell acute lymphoblastic leukemia (B-ALL) significantly improves with adequate exposure (as Area Under the Curve: AUC_0-inf or AUC) to fludarabine (FLU) during lymphodepletion prior to chimeric antigen receptor (CAR) T-cell infusion. A cumulative AUC of FLU exceeding 14 mgxh/L is considered optimal, making therapeutic drug monitoring (TDM) crucial for personalized dosing and prolonged CAR T-cell persistence.

Research design and methods: We developed and validated an European Medicines Agency (EMA)-compliant high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) for FLU TDM, based on similar and published methods, to support clinicians during the patient conditioning phase. All EMA acceptance criteria were met.

Results: Analysis of real plasma samples revealed unexpectedly high FLU concentrations and, consequently, an excessively high AUC. Analysis of plasma samples from a child receiving FLU therapy highlighted the necessity for accurate drug response verification in real matrices. Preparation of quality controls in FLU-free plasma consistently failed without preliminary dilution. Analysis of internal standard range variation enabled more precise and accurate analyses.

Conclusions: This experience highlights the importance of complementing standard validation tests with analyses in each patient's plasma, collected immediately before FLU infusion, to ensure reliable TDM to support CAR T-cell therapy.

Aim: Ischemic stroke (IS) occurs when a clot obstructs cerebral blood flow, leading to potentially irreversible damage. Metabolites, as the end products of cellular metabolism, can reflect pathological changes in the body. This study aimed to characterize serum metabolic alterations associated with acute ischemic stroke.

Method: Participants were divided into discovery and validation sets, each comprising healthy controls and IS patients. In the discovery set, untargeted serum metabolomic profiling was performed using liquid chromatography - tandem mass spectrometry. Metabolites showing significant intergroup differences were selected as biomarker candidates and quantitatively validated in the independent set. Diagnostic performance was assessed using receiver operating characteristic (ROC) curve analysis.

Results: Three metabolites - cytidine, decanoyl-L-carnitine, and 2-hydroxyestradiol - were identified as significantly altered in IS patients. Cytidine and 2-hydroxyestradiol demonstrated excellent diagnostic performance (AUCs of 0.968 and 0.929, respectively), while decanoyl-L-carnitine showed good performance (AUC = 0.808).

Conclusion: The three metabolites exhibit significant alterations in acute ischemic stroke patients and provide insight into disease-associated metabolic changes. Their validation lays the groundwork for future exploratory studies investigating potential biomarkers.