The use of liquid chromatography-mass spectrometry for quantitative large-molecule bioanalysis has increased substantially over the past decade, driven by its ability to address analytical challenges that may be difficult to resolve using traditional ligand-binding assays. In this European Bioanalysis Forum perspective, seven case studies from pharmaceutical companies and contract research organizations illustrate the fit-for-purpose application of LC-MS across different stages of drug development. These include isoform-specific quantification, subclass-resolved immunoglobulin profiling, characterization of post-translational modifications, assessment of in vivo biotherapeutic integrity, and multiplexed analysis in complex biological matrices. Collectively, the examples highlight how LC-MS can support selectivity, structural insight, and analytical flexibility when aligned with the specific context of use. The EBF aims to foster continued scientific exchange by encouraging the sharing of such case studies across its community.

BACKGROUND AND PURPOSE:The chemokine receptor CCR6 guides pathogenic T17 cells, implicated in autoimmune diseases including psoriasis, to sites of inflammation via the chemokine CCL20. Therefor, pharmacological inhibition of CCR6+ immune cell migration provides a novel therapeutic approach. Translatability of such an intervention has not yet been assessed in detail. We evaluated the translatability of the Aldara® mouse model induced skin inflammation to psoriasis, with particular focus on immune cell trafficking and assessed the efficacy of IDOR-1117-2520, a highly selective, potent and orally available CCR6 small inhibitor. EXPERIMENTAL APPROACH:Effects of IDOR-1117-2520 were investigated in the Aldara® and IL23 mouse models of skin inflammation using flow cytometry, RNA sequencing and transcriptome-based cell type deconvolution approaches to characterise immune cell migration patterns. These results were compared to human psoriasis transcriptomics data. KEY RESULTS:IDOR-1117-2520 dose dependently reduced infiltration of CCR6+ immune cells into inflamed skin, and was equally efficacious as IL-17 and IL-23 inhibition in models of skin inflammation. Pathway analysis showed molecular similarities in the immune response between human psoriasis and the Aldara® mouse model. IL-17/IL-23 pathway genes were expressed in both human psoriasis and the mouse model. CCR6 inhibition modulated multiple pathways associated with inflammation beyond the proximal IL-17/IL-23 pathway. A chemokine-chemokine receptor interaction map implicated CCL20-CCR6 as the dominant axis in recruiting pathogenic T17 cells in both the model and in human psoriasis. CONCLUSION AND IMPLICATIONS:IDOR-1117-2520 could provide a promising novel targeted approach to treating psoriasis and, potentially, other autoimmune diseases involving the CCR6/CCL20 axis and the IL-17/IL-23 pathway. IDOR-1117-2520 is currently being evaluated in a clinical phase 1 trial (ISRCTN28892128).

The European Bioanalysis Forum has observed increasing misclassification of pharmacokinetic, anti-drug antibody, and biomarker research assays not used for patient management under the European Union's In Vitro Diagnostic Regulation, despite their non-diagnostic intent in early clinical development. This misinterpretation, fueled by ambiguous protocol language, limited cross-functional awareness, and inconsistent national implementation, is leading to unnecessary delays in clinical trials and increased and non-added value regulatory burden. Through a structured evaluation involving a focus workshop and regional roadshows, the European Bioanalysis Forum identified some manageable origins of the issue and its operational consequences. This recommendation paper outlines these observations and wants to propose a pragmatic path forward. This includes clearer regulatory guidance to exempt noncommercial, non-diagnostic assays from In Vitro Diagnostic Regulation when not developed or intended as registered diagnostics. We also highlight the importance of stakeholder education and coordinated regulatory dialogue. These steps aim to preserve the regulator's intent of patient protection while enabling timely and efficient clinical research.

The European Bioanalysis Forum, alongside key industry stakeholders, has been driving the discussions around the implementation of context-of use for biomarker assays to ensure that these assays are validated appropriately depending on their purpose. Insights into understanding why the implementation of context-of-use in assay strategies has also shown that the key stakeholder, or requester for the biomarker data, is responsible for providing the context-of-use statement for all biomarker assay requests. Experts from across the industry haves repeatedly sought a cross-industry recommended format in which the context-of-use statement could be provided. In this manuscript, the European Bioanalysis Forum suggests a format for this.

Hypothesis: Endothelin (ET)-1 is upregulated in hypertension and especially resistant forms of hypertension. As ET-1 is a paracrine hormone involved in renal organ damage, we investigated the correlation between eGFR and activity of the ET system in the resistant hypertensive population of the PRECISION trial (NCT03541174), in which aprocitentan, a dual endothelin ET A /ET B receptor antagonist, significantly reduced blood pressure and UACR. To assess activity of the ET system, ET-1 and CT-proET-1, a stable peptide and biomarker for ET-1 production, were measured in plasma. Methods: From plasma collected at screening from the future randomized patients in PRECISION, ET-1 (n=688) and CT-proET-1 (n=100, randomly selected) were measured by ELISA and LC-MS-based assays respectively. Baseline levels of CT-proET-1 were determined on pooled plasma from healthy adults (n=70). Pearson correlation coefficient was determined between biomarkers of the ET system and eGFR. Results: CT-proET-1 concentrations measured in patients with resistant hypertension were higher than concentrations from healthy subjects (83 vs 55 pM). In patients with an eGFR≥60 mL/min/1.73m 2 , mean CT-proET-1 concentration was 75 pM [95% CI 70-81; n=77] as compared to 116 pM [95% CI 95-140 n=23] in patients with eGFR<60 mL/min/1.73m 2 . In these two subgroups, mean ET-1 concentrations were 1.90 ng/L [95% CI 1.85-1.95; n=531] and 2.10 ng/L [95% CI 1.99-2.22; n=157] for patients with an eGFR≥60 and eGFR<60 mL/min/1.73m 2 respectively. There was an inverse correlation between CT-proET-1 and eGFR (r=-0.61, p<0.0001) and a less pronounced correlation between ET-1 and eGFR (r=-0.11, p=0.0027) (see Figure ). Conclusions: In patients with resistant hypertension, there was a strong inverse correlation between plasma CT-proET-1 and renal function, suggesting the role of the ET system not only in hypertension but also in renal damage. These data demonstrate that CT-proET-1 is a sensitive biomarker of the ET system, reflecting the production of ET-1.

Hypothesis: Dual endothelin (ET) ET A /ET B receptor antagonist aprocitentan significantly reduced blood pressure in patients with resistant hypertension, who were still hypertensive despite standard background anti-hypertensive drugs including a diuretic, an ARB and a CCB (PRECISION trial, NCT03541174). The effect of aprocitentan in this population, characterized with low renin and elevated aldosterone levels, was assessed on a panel of plasma biomarkers: ET-1, CT-proET-1 (a stable peptide and biomarker for ET-1 production), renin, aldosterone. Methods: From the 730 randomized patients in PRECISION, ET-1 (n=700), CT-proET-1 (n=100, randomly selected), immunoreactive renin (n=679) and aldosterone (n=703) were measured in plasma samples collected at randomization (baseline) and after 4 weeks for either placebo, aprocitentan 12.5 or 25 mg. Results: Four-week placebo treatment had no effect on the studied biomarkers compared to baseline. Treatment with aprocitentan 12.5 and 25 mg increased plasma ET-1 by +34% and +53% and CT-proET-1 by +38% and +47% vs. baseline respectively. Aprocitentan had no effect on renin levels (p>0.05) while decreasing plasma aldosterone by -17% and -24% at the 12.5 and 25 mg dose respectively. Importantly, the reduction in aldosterone did not result in a change in kalemia (see Table ). Conclusions: Aprocitentan caused reactive dose-dependent increases in plasma CT-proET-1 and ET-1, resulting from effective ET receptor blockade. The increase in CT-proET-1 may indicate a compensatory increase in ET-1 de novo synthesis, while the increase in ET-1 may reflect a reduced clearance due to blockade of ET B and increased production. The decrease of aldosterone by aprocitentan confirms the secretagogue role of ET-1 on aldosterone production by adrenal cortical cells via both ET A and ET B receptors This decrease of aldosterone was observed on top of the known decrease induced by an ARB but did not induce hyperkalemia nor aggravation of renal function.

Abstract CXCL12 acts as a chemoattractant by binding to the receptor CXCR4. The (atypical) chemokine receptor ACKR3 (CXCR7) scavenges CXCL12. Antagonism of ACKR3 thus leads to an increase in CXCL12 concentrations that has been used as a pharmacodynamic biomarker in healthy adults. Increased CXCL12 concentrations have also been linked to repair mechanisms in human diseases and mouse models. To date, CXCL12 concentrations have typically been quantified using antibody‐based assays with overlapping or unclear specificity for the various CXCL12 isoforms (α, β, and γ) and proteoforms. Only the N‐terminal full‐length CXCL12 proteoform is biologically active and can engage CXCR4 and ACKR3, but this proteoform could so far not be quantified in healthy adults. Here, we describe a new and fit‐for‐purpose validated immunoaffinity mass spectrometry (IA‐MS) assay for specific measurement of five CXCL12α proteoforms in human plasma, including the biologically active CXCL12α proteoform. This biomarker assay was used in a phase I clinical study with the ACKR3 antagonist ACT‐1004‐1239. In placebo‐treated healthy adults, 1.0 nM total CXCL12α and 0.1 nM biologically active CXCL12α was quantified. The concentrations of both proteoforms increased up to two‐fold in healthy adults compared to placebo following drug administration. At all dose levels, 10% of the CXCL12α was the biologically active proteoform and the simultaneous increase of all proteoforms suggests that a new steady state has been reached 24 h following dosing. Hence, this IA‐MS biomarker assay can be used to specifically measure active CXCL12 proteoform concentrations in clinical trials to demonstrate target engagement and correlate with clinical outcomes.

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.

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.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

Mass spectrometry-based proteomics has already contributed greatly to systems biology studies and its role in systems biology research is likely to grow in the next few years. Its unique capability of quantifying proteins and protein modifications at large scale and throughput make it a prime technology in systems biology studies, specifically those focused on complex biological processes and diseases. In this article, we first introduce the motivation and challenges of using proteomics in systems biology studies. Second, we give an overview of the most common methods to measure and quantify proteins using liquid-chromatography coupled tandem mass spectrometry. In the third section, we highlight how mass spectrometry contributes to generating functionally highly relevant proteomic information that goes beyond the identity and quantity of constituent proteins (i.e., mapping the proteoforms, mapping the interactions, and relating the proteome to the genome), and then we focus on three exemplary biological processes to show how mass spectrometry-based studies contributed to increasing our understanding of them.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

Background Recent efforts have described the evolution of glioblastoma from initial diagnosis to post-treatment recurrence on a genomic and transcriptomic level. However, the evolution of the proteomic landscape is largely unknown. Methods Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used to characterize the quantitative proteomes of two independent cohorts of paired newly diagnosed and recurrent glioblastomas. Recurrence-associated proteins were validated using immunohistochemistry and further studied in human glioma cell lines, orthotopic xenograft models, and human organotypic brain slice cultures. External spatial transcriptomic, single-cell, and bulk RNA sequencing data were analyzed to gain mechanistic insights. Results Although overall proteomic changes were heterogeneous across patients, we identified BCAS1, INF2, and FBXO2 as consistently upregulated proteins at recurrence and validated these using immunohistochemistry. Knockout of FBXO2 in human glioma cells conferred a strong survival benefit in orthotopic xenograft mouse models and reduced invasive growth in organotypic brain slice cultures. In glioblastoma patient samples, FBXO2 expression was enriched in the tumor infiltration zone and FBXO2-positive cancer cells were associated with synaptic signaling processes. Conclusions These findings demonstrate a potential role of FBXO2-dependent glioma-microenvironment interactions to promote tumor growth. Furthermore, the published datasets provide a valuable resource for further studies.

Protein complexes are responsible for the enactment of most cellular functions. For the protein complex to form and function, its subunits often need to be present at defined quantitative ratios. Typically, global changes in protein complex composition are assessed with experimental approaches that tend to be time consuming. Here, we have developed a computational algorithm for the detection of altered protein complexes based on the systematic assessment of subunit ratios from quantitative proteomic measurements. We applied it to measurements from breast cancer cell lines and patient biopsies and were able to identify strong remodeling of HDAC2 epigenetic complexes in more aggressive forms of cancer. The presented algorithm is available as an R package and enables the inference of changes in protein complex states by extracting functionally relevant information from bottom-up proteomic datasets.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α-smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology-a strategy that is based on perturbing primary tumor cells from cancer patients-could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC.

Complex traits are characterized by multiple genes and variants acting simultaneously on a phenotype. However, studying the contribution of individual pairs of genes to complex traits has been challenging since human genetics necessitates very large population sizes, while findings from model systems do not always translate to humans. Here, we combine genetics with combinatorial RNAi (coRNAi) to systematically test for pairwise additive effects (AEs) and genetic interactions (GIs) between 30 lipid genome-wide association studies (GWAS) genes. Gene-based burden tests from 240,970 exomes show that in carriers with truncating mutations in both, APOB and either PCSK9 or LPL ("human double knock-outs") plasma lipid levels change additively. Genetics and coRNAi identify overlapping AEs for 12 additional gene pairs. Overlapping GIs are observed for TOMM40/APOE with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate plasma and cellular lipid levels primarily via AEs and nominates putative drug target pairs for improved lipid-lowering combination therapies.

SUMMARY Genetic interactions (GIs), the joint impact of different genes or variants on a phenotype, are foundational to the genetic architecture of complex traits. However, identifying GIs through human genetics is challenging since it necessitates very large population sizes, while findings from model systems not always translate to humans. Here, we combined exome-sequencing and genotyping in the UK Biobank with combinatorial RNA-interference (coRNAi) screening to systematically test for pairwise GIs between 30 lipid GWAS genes. Gene-based protein-truncating variant (PTV) burden analyses from 240,970 exomes revealed additive GIs for APOB with PCSK9 and LPL , respectively. Both, genetics and coRNAi identified additive GIs for 12 additional gene pairs. Overlapping non-additive GIs were detected only for TOMM40 at the APOE locus with SORT1 and NCAN . Our study identifies distinct gene pairs that modulate both, plasma and cellular lipid levels via additive and non-additive effects and nominates drug target pairs for improved lipid-lowering combination therapies.

Predicting how a system behaves under changing conditions is an essential component of science and engineering. The ability to make accurate predictions about the system indicates that it is well understood and provides the opportunity to simulate the response to conditions that would be empirically difficult or impossible to test. In the life sciences, the term systems biology was introduced to articulate the notion that the molecular and phenotypic response of a cell or organism to perturbations is the result of interplay of a multitude of molecules. The ability to predict the behavior of such complex molecular systems remains challenging and inevitably requires the involvement of different types of models and data that support them. In this article, we discuss a range of data-driven models that have proven particularly useful for predicting the behavior of biological systems at different levels of complexity and the matching data generation methods that support them. We specifically focus on predictions based on protein or proteome data generated by mass spectrometry. We describe three case studies that represent frequently encountered situations in systems biology.