Development and characterization of a 3D in vitro model mimicking acneic skin.

Acne is an inflammatory skin disease of the pilosebaceous unit, involving four essential factors: hyperseborrhoea combined to a modification of sebum composition, colonization by Cutibacterium (C.) acnes, hyperkeratinization and secreted inflammation. Understanding and mimicking compromised skin is essential to further develop appropriate therapeutic solutions. This study aimed to develop new in vitro 3D models mimicking acneic skin, by combining two main factors involved in the physiopathology, namely, altered sebum composition and C. acnes invasion. Normal human keratinocytes were first used to generate reconstructed human epidermis (RHE) that were then left untreated (control) or treated topically with a combination of both peroxidized squalene and C. acnes cultures. Once validated, this model considered relevant to mimic acneic skin, was further improved by using different phylotypes of C. acnes strains specifically isolated from healthy and acneic patients. While both phylotypes IB and II did not significantly alter RHE, C. acnes IA1 strains induce major acneic skin hallmarks such as hyperkeratinization, secreted inflammation and altered barrier function. Interestingly, these results are obtained independently of the origin of IA1 phylotypes (acneic vs. healthy patient), thus suggesting a role of the ecosystem in controlling C. acnes virulence in healthy skin. In conclusion, by combining two major factors involved in the physiopathology of acne, we (1) succeeded to design in vitro 3D models mimicking this skin disorder and (2) highlighted how C. acnes phylotypes can have an impact on epidermal physiology. These relevant models will be suitable for the substantiation of therapeutic molecules dedicated to acne treatment.

Safety, pharmacokinetics and pharmacodynamics of BI 655064 in phase 1 clinical trials in healthy Chinese and Japanese subjects.

AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of BI 655064 in healthy Chinese and Japanese subjects after administration of single doses of 80-240 mg and multiple dosing of 240 mg once weekly over 4 weeks. METHODS: Two phase 1, double-blind, placebo-controlled studies were conducted (single-rising doses of BI 655064 in Chinese/Japanese male subjects [n = 12 per BI 655064 dose group] or repeated 240 mg BI 655064 in Chinese male subjects [n = 9]). Plasma samples were collected to investigate BI 655064 pharmacokinetics, pharmacodynamics (CD40 receptor occupancy [RO]) and immunogenicity, along with the safety and tolerability of BI 655064. RESULTS: BI 655064 showed good overall tolerability following single-dose administration of 80-240 mg and repeated administration of 240 mg BI 655064 over 4 weeks. More Chinese subjects reported adverse events compared with Japanese subjects following single-dose administration (59.4% vs 3.1%). BI 655064 exhibited nonlinear, saturable kinetics, with higher doses resulting in slower apparent clearance (0.514-0.713 mL min-1 ), and disproportionately higher total exposure (AUC0-inf ; 5610-7780 µg·h mL-1 ) and maximum plasma concentration (15 700-21 300 ng mL-1 ) with 240 mg BI 655064. Ninety percent inhibition of CD40 RO was achieved with doses ≥120 mg, and a direct relationship between BI 655064 plasma concentration and inhibition of CD40 RO was observed. Most subjects had a positive treatment-emergent antidrug antibody response. CONCLUSIONS: BI 655064 pharmacokinetic and safety profiles in East Asian male subjects were consistent with those observed in a Western population. No adjustments in the BI 655064 dosing recommendations are warranted for future clinical trials.

Current strategies in the non-clinical safety assessment of biologics: New targets, new molecules, new challenges.

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing-based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Council for Harmonization (ICH) S1, S3a, S5, S9 and S11 and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species.

Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor.

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

Cytokine release: A workshop proceedings on the state-of-the-science, current challenges and future directions.

In October 2013, the International Life Sciences Institute - Health and Environmental Sciences Institute Immunotoxicology Technical Committee (ILSI-HESI ITC) held a one-day workshop entitled, "Workshop on Cytokine Release: State-of-the-Science, Current Challenges and Future Directions". The workshop brought together scientists from pharmaceutical, academic, health authority, and contract research organizations to discuss novel approaches and current challenges for the use of in vitro cytokine release assays (CRAs) for the identification of cytokine release syndrome (CRS) potential of novel monoclonal antibody (mAb) therapeutics. Topics presented encompassed a regulatory perspective on cytokine release and assessment, case studies regarding the translatability of preclinical cytokine data to the clinic, and the latest state of the science of CRAs, including comparisons between mAb therapeutics within one platform and across several assay platforms, a novel physiological assay platform, and assay optimization approaches such as determination of FcR expression profiles and use of statistical tests. The data and approaches presented confirmed that multiple CRA platforms are in use for identification of CRS potential and that the choice of a particular CRA platform is highly dependent on the availability of resources for individual laboratories (e.g. positive and negative controls, number of human blood donors), the assay through-put required, and the mechanism-of-action of the therapeutic candidate to be tested. Workshop participants agreed that more data on the predictive performance of CRA platforms is needed, and current efforts to compare in vitro assay results with clinical cytokine assessments were discussed. In summary, many laboratories continue to focus research efforts on the improvement of the translatability of current CRA platforms as well explore novel approaches which may lead to more accurate, and potentially patient-specific, CRS prediction in the future.

2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. 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 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th 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 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Antigen is required for maturation and activation of pathogenic anti-DNA antibodies and systemic inflammation.

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies and systemic inflammation that results in part from dendritic cell activation by nucleic acid containing immune complexes. There are many mouse models of lupus, some spontaneous and some induced. We have been interested in an induced model in which estrogen is the trigger for development of a lupus-like serology. The R4A transgenic mouse expresses a transgene-encoded H chain of an anti-DNA Ab. This mouse maintains normal B cell tolerance with deletion of high-affinity DNA-reactive B cells and maturation to immunocompetence of B cells making nonglomerulotropic, low-affinity DNA-reactive Abs. When this mouse is given estradiol, normal tolerance mechanisms are altered; high-affinity DNA-reactive B cells mature to a marginal zone phenotype, and the mice are induced to make high titers of anti-DNA Abs. We now show that estradiol administration also leads to systemic inflammation with increased B cell-activating factor and IFN levels and induction of an IFN signature. DNA must be accessible to B cells for both the production of high-affinity anti-DNA Abs and the generation of the proinflammatory milieu. When DNase is delivered to the mice at the same time as estradiol, there is no evidence for an abrogation of tolerance, no increased B cell-activating factor and IFN, and no IFN signature. Thus, the presence of autoantigen is required for positive selection of autoreactive B cells and for the subsequent positive feedback loop that occurs secondary to dendritic cell activation by DNA-containing immune complexes.

Trends in industrialization of biotherapeutics: a survey of product characteristics of 89 antibody-based biotherapeutics.

There is considerable interest in the pharmaceutical industry toward development of antibody-based biotherapeutics because they can selectively bind diverse receptors and often possess desirable pharmacology. Here, we studied product characteristics of 89 marketed antibody-based biotherapeutics that were approved from 1986 to mid-2020 by gathering publicly available information. Our analyses revealed major trends in their emergence as the best-selling class of pharmaceuticals. Early on, most therapeutic monoclonal antibodies were developed to treat cancer, with CD20 being the most common target. Thanks to industrialization of antibody manufacturing technologies, their use has now blossomed to include 15 different therapeutic areas and nearly 60 targets, and the field is still growing! Drug manufacturers are solidifying their choices regarding types of antibodies and their molecular formats. IgG1 kappa continues to be the most common molecular format among marketed antibody-based biotherapeutics. Most antibody-based biotherapeutics approved since 2015 are either humanized or fully human, but the data we collected do not show a direct correlation between humanness and reported incidence of anti-drug antibodies. Furthermore, there have also been improvements in terms of drug product stability and high concentration liquid formulations suitable for subcutaneous route of administration, which are being approved more often in recent years. These improvements, however, have not been uniformly adopted across all therapeutic areas, suggesting that multiple options for drug product development are being used to serve diverse therapeutic purposes. Insights gained from this analysis may help us devise better end-to-end antibody-based biotherapeutic drug discovery and development strategies.

Therapeutic Protein Drug Interactions: A White Paper From the International Consortium for Innovation and Quality in Pharmaceutical Development.

Typically, therapeutic proteins (TPs) have a low risk for eliciting meaningful drug interactions (DIs). However, there are select instances where TP drug interactions (TP-DIs) of clinical concern can occur. This white paper discusses the various types of TP-DIs involving mechanisms such as changes in disease state, target-mediated drug disposition, neonatal Fc receptor (FcRn), or antidrug antibodies formation. The nature of TP drug interaction being investigated should determine whether the examination is conducted as a standalone TP-DI study in healthy participants, in patients, or assessed via population pharmacokinetic analysis. DIs involving antibody-drug conjugates are discussed briefly, but the primary focus here will be DIs involving cytokine modulation. Cytokine modulation can occur directly by certain TPs, or indirectly due to moderate to severe inflammation, infection, or injury. Disease states that have been shown to result in indirect disease-DIs that are clinically meaningful have been listed (i.e., typically a twofold change in the systemic exposure of a coadministered sensitive cytochrome P450 substrate drug). Type of disease and severity of inflammation should be the primary drivers for risk assessment for disease-DIs. While more clinical inflammatory marker data needs to be collected, the use of two or more clinical inflammatory markers (such as C-reactive protein, albumin, or interleukin 6) may help broadly categorize whether the predicted magnitude of inflammatory disease-DI risk is negligible, weak, or moderate to strong. Based on current knowledge, clinical DI studies are not necessary for all TPs, and should no longer be conducted in certain disease patient populations such as psoriasis, which do not have sufficient systemic inflammation to cause a meaningful indirect disease-DI.

Current nonclinical approaches for immune assessments of immuno-oncology biotherapeutics.

Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.

Nonclinical pharmacokinetics and biodistribution of VSV-GP using methods to decouple input drug disposition and viral replication.

Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.

Genome sequence of the probiotic strain Bifidobacterium animalis subsp. lactis CNCM I-2494.

Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.

Differential roles of estrogen receptors α and ß in control of B-cell maturation and selection.

It is clear that estrogen can accelerate and exacerbate disease in some lupus-prone mouse strains. It also appears that estrogen can contribute to disease onset or flare in a subset of patients with lupus. We have previously shown estrogen alters B-cell development to decrease lymphopoiesis and increase the frequency of marginal zone B cells. Furthermore, estrogen diminishes B-cell receptor signaling and allows for the increased survival of high-affinity DNA-reactive B cells. Here, we analyze the contribution of estrogen receptor α or ß engagement to the altered B-cell maturation and selection mediated by increased exposure to estrogen. We demonstrate that engagement of either estrogen receptor α or ß can alter B-cell maturation, but only engagement of estrogen receptor α is a trigger for autoimmunity. Thus, maturation and selection are regulated differentially by estrogen. These observations have therapeutic implications.

Using multiple platforms for critical reagents selection process to support pharmacokinetic ligand-binding assay development.

We have evaluated the utility of epitope binning on biolayer interferometry (BLI) as a strategy to funnel the selection of candidate pairs suitable for pharmacokinetic assay development. Totally, 8 anti-Idiotypic monoclonal antibodies in 64 possible combinations were tested by BLI, ELISA and Gyrolab®. Two epitope binning approaches were utilized, in-tandem and classic sandwich. Both formats identified four mutually exclusive bins providing 31 and 25 possible antibody pair combinations, respectively. In contrast, the ELISA and Gyrolab yielded 18 and 9 positive pairs, respectively, with only a partial correlation to the BLI results. Several positive pairs by ELISA and Gyrolab, screened negative by BLI. Just over half of the pairs predicted by BLI were positive on ELISA and less than a quarter were positive on Gyrolab. This evaluation showed, in our case, that BLI was limited in its ability to predict candidate pairs that would be successful in pharmacokinetic method development.

Critical reagent generation, characterization, handling and storage workflows: impact on ligand binding assays.

The foundation of pharmacokinetics and antidrug antibodies assay robustness relies on the use of high-quality reagents. Over the past decade, there has been increasing interest within the pharmaceutical industry, as well as regulators, on defining best practices and scientific approaches for generation, characterization and handling of critical reagents. In this review, we will discuss current knowledge and practices on critical reagent workflows and state-of-the-art approaches for characterization, generation, stability and storage and how each of these steps can impact ligand-binding assay robustness.

Lay abstract A critical part of clinical development for new biologic drugs is the use of tests known as ligand-binding assay. These assays must be able to accurately measure drug levels and to assess if the biologic drug interacts with the immune system in patients. In order to support patient efficacy and safety, scientists must use state-of-the-art approaches to develop and identify specific reagents for each new biologic drug. This review aims to cover all key steps that are needed to support the quality and performance of the unique components of ligand-binding assays from the beginning of assay development and throughout the entire life-cycle of the biologic drug.

An optimally designed anti-human CD40 antibody with potent B cell suppression for the treatment of autoimmune diseases.

Antibodies targeting the CD40-CD40L pathway have great potential for treating autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), and inflammatory bowel diseases (IBD). However, in addition to the known difficulty in generating a purely antagonistic CD40 antibody, the presence of CD40 and CD40L on platelets creates additional unique challenges for the safety, target coverage, and clearance of antibodies targeting this pathway. Previously described therapeutic antibodies targeting this pathway have various shortcomings, and the full therapeutic potential of this axis has yet to be realized. Herein, we describe the generation and characterization of BI 655064, a novel, purely antagonistic anti-CD40 antibody that potently neutralizes CD40-CD40L-dependent B-cell stimulation without evidence of impacting platelet functions. This uniquely optimized antibody targeting a highly challenging pathway was obtained by applying stringent functional and biophysical criteria during the lead selection process. BI 655064 has favorable target-mediated drug disposition (TMDD)-saturation pharmacokinetics, consistent with that of a high-quality therapeutic monoclonal antibody.

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

Selective regulation of autoreactive B cells by FcgammaRIIB.

FcgammaRIIB is an inhibitory receptor which plays a role in limiting B cell and DC activation. Since FcgammaRIIB is known to dampen the signaling strength of the BCR, we wished to determine the impact of FcgammaRIIB on the regulation of BCRs which differ in their affinity for DNA. For these studies, FcgammaRIIB deficient BALB/c mice were bred with mice expressing the transgene-encoded H chain of the R4A anti-DNA antibody which gives rise to BCRs which express high, low or no affinity for DNA. The deletion of FcgammaRIIB in R4A BALB/c mice led to an alteration in the B cell repertoire, allowing for the expansion and activation of high affinity DNA-reactive B cells. By 6-8 months of age, R4A x FcgammaRIIB-/- BALB/c mice spontaneously developed anti-DNA antibody titers. These mice also displayed an induction of IFN-inducible genes and an elevation in levels of the B cell survival factor, BAFF. These data demonstrate that FcgammaRIIB preferentially limits activation of high affinity autoreactive B cells and can influence the activation of DC through an immune complex-mediated mechanism.

Retrospective analysis of model-based predictivity of human pharmacokinetics for anti-IL-36R monoclonal antibody MAB92 using a rat anti-mouse IL-36R monoclonal antibody and RNA expression data (FANTOM5).

Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1 κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.