Serum proteomics reveals APOE dependent and independent protein signatures in Alzheimer's disease.

The current demand for early intervention, prevention, and treatment of late onset Alzheimer's disease (LOAD) warrants deeper understanding of the underlying molecular processes which could contribute to biomarker and drug target discovery. Utilizing high-throughput proteomic measurements in serum from a prospective population-based cohort of older adults (n = 5,294), we identified 303 unique proteins associated with incident LOAD (median follow-up 12.8 years). Over 40% of these proteins were associated with LOAD independently of APOE-ε4 carrier status. These proteins were implicated in neuronal processes and overlapped with protein signatures of LOAD in brain and cerebrospinal fluid. We found 17 proteins which LOAD-association was strongly dependent on APOE-ε4 carrier status. Most of them showed consistent associations with LOAD in cerebrospinal fluid and a third had brain-specific gene expression. Remarkably, four proteins in this group (TBCA, ARL2, S100A13 and IRF6) were downregulated by APOE-ε4 yet upregulated as a consequence of LOAD as determined in a bi-directional Mendelian randomization analysis, reflecting a potential response to the disease onset. Accordingly, the direct association of these proteins to LOAD was reversed upon APOE-ε4 genotype adjustment, a finding which we replicate in an external cohort (n = 719). Our findings provide an insight into the dysregulated pathways that may lead to the development and early detection of LOAD, including those both independent and dependent on APOE-ε4. Importantly, many of the LOAD-associated proteins we find in the circulation have been found to be expressed - and have a direct link with AD - in brain tissue. Thus, the proteins identified here, and their upstream modulating pathways, provide a new source of circulating biomarker and therapeutic target candidates for LOAD.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

Signature peptide selection workflow for biomarker quantification using LC-MS-based targeted proteomics.

In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC-MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.

Development of a UPLC-MRM-based targeted proteomic method to profile subcellular organelle marker proteins from human liver tissues.

Subcellular organelles have long been an interest in biochemical research and drug development as the isolation of those organelles can help to probe protein functions and elucidate drug disposition within the cell. Usually, the purity of isolated subcellular organelle fractions was determined using immunoblot analysis of subcellular organelle marker proteins, which can be labor-intensive and lack reproducibility due to antibody batch-to-batch variability. As such, a higher throughput and more robust method is needed. Here, a UPLC-MRM-based targeted proteomic method was developed for a panel of human organelle marker proteins and used to profile a series of sucrose fractions isolated from the protein extract of human liver tissues. The method was validated by comparing to the traditional immunoblot and determining subcellular localization of three case study proteins (CYP3A4, FcRn, and ß2M) pertaining to the disposition of small molecule and biologic drugs. All three case study proteins were co-enriched with their corresponding subcellular protein marker, and complete recoveries were achieved from isolated fractions. This newly developed MRM method for the panel of human organelle marker proteins can potentially accelerate future intracellular drug disposition analysis and facilitate subcellular organelle quality assessment.

Comparison of three parallelism assessment methods of biomarker quantification by LC-MS/MS: a case study of the bioanalysis of creatinine in human urine samples.

Background: Currently, no regulatory guidelines are available for parallelism assessment for LC-MS biomarker quantification. Spike recovery, standard addition and dilutional linearity are recommended with no mention of the implications of applying these approaches. Results: Here, using human urine creatinine, the authors compared spike recovery and standard addition in LC-MS biomarker quantification, and evaluated a new hybrid approach: parallelism QCs. The authors drew different conclusions based on which approach was used (<15% cutoff). Conclusion: Current recommended approaches may lead to different conclusions and are not equivalent and interchangeable. The authors recommend that standard addition should be the universal 'go-to' method for LC-MS biomarker parallelism assessment; parallelism QCs, which consider the total concentration as the theoretical value, can be used if the authentic matrix is limited.

Fit-for-purpose LC-MS/MS quantification of leukotoxin and leukotoxin diol in mouse plasma without sample pre-concentration.

LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes. Additionally, omitting pre-concentration steps streamlines sample processing, which can now be completed in under 10 min. This method can be used to quickly answer if the ratio of LTX to LTXdiol changes with the dose of the therapeutic drug so this could be used as a potential biomarker for correlating PK/PD effects. No extensive assay characterization was performed before application to an exploratory in-life study. Basal levels of LTX and LTXdiol in plasma were quantified by LC-MRM across 10 individual mice, and the average signal-to-noise was 36 for LTX and 3039 for LTXdiol, with CVs of 29.4% and 15.2%, respectively. Addition of LTX and LTXdiol reference standard at 5, 25, and 75 ng/mL into pooled mouse plasma was quantifiable within 30% relative error using a surrogate matrix calibration curve ranging from 0.8 to 200 ng/mL. The average ratio of LTX to LTXdiol across the 10 mice was 0.32, consistent with previous reports. Finally, the method was applied to a mouse PK/PD study to monitor LTX/LTXdiol kinetics after a single oral dose of a soluble epoxide hydrolase inhibitor. The mean plasma ratio of LTX to LTXdiol increased up to 10-fold by 3 h post-dose followed by a decrease to near pre-dose levels by 24 h, consistent with transient inhibition of sEH-mediated conversion of LTX to LTXdiol. The method improvements described here will make subsequent quantification of LTX and LTXdiol in mouse studies significantly easier.

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models.

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and ß2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and ß2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and ß2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic ß2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and ß2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and ß2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.