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The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain.
No full textAssessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs
Considerations for the Terminal Sterilisation of Oligonucleotide Drug Products
No full textA primary function of the parenteral drug product manufacturing process is to ensure sterility of the final product. The two most common methods for sterilizing parenteral drug products are terminal sterilisation (TS), whereby the drug product is sterilized in the final container following filling and finish, and membrane sterilisation, whereby the product stream is sterilized by membrane filtration and filled into pre-sterilized containers in an aseptic processing environment. Though TS provides greater sterility assurance than membrane sterilisation and aseptic processing, not all drug products are amenable to TS processes, which typically employ heat treatment or exposure to ionizing radiation.
Oligonucleotides represent an emerging class of therapeutics that have great potential to treat a broad range of indications, including previously undruggable targets. Due to their size, structural complexity, and relative lack of governing regulations, there are several challenges in drug development that are unique for oligonucleotides. This exceptionality justifies a focused assessment of traditional chemistry, manufacturing, and control strategies prior to their adoption. In this article, we review the current state of sterile oligonucleotide drug product processing, highlight key aspects to consider when assessing options for product sterilisation, and provide recommendations to aid in the successful evaluation and development of TS processes. We also explore the current regulatory expectations and provide our interpretation as it pertains to oligonucleotide drug product
The evolving role of investigative toxicology in the pharmaceutical industry.
No full textFor decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies
An Intra-Company Analysis of Inherent Particles in Biologicals Shapes the Protein Particle Mitigation Strategy Across Development Stages.
No full textTo better understand protein aggregation and inherent particle formation in the biologics pipeline at Novartis, a cross-functional team collected and analyzed historical protein particle issues. Inherent particle occurrences from the past 10 years were systematically captured in a protein particle database. Where the root cause was identified, a number of product attributes (such as development stage, process step, or protein format) were trended. Several key themes were revealed: 1) there was a higher propensity for inherent particle formation with non-mAbs than with mAbs; 2) the majority of particles were detected following manufacturing at scale, and were not predicted by the small-scale studies; 3) most issues were related to visible particles, followed by subvisible particles; 4) 50% of the issues were manufacturing related. These learnings became the foundation of a particle mitigation strategy across development and technical transfer, and resulted in a set of preventive actions. Overall, this study provides further insight into a recognized industry challenge and hopes to inspire the biopharmaceutical industry to transparently share their experiences with inherent particles formation
Interlaboratory Variability in the Madin-Darby Canine Kidney Cell Proteome.
No full textMadin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory
Comparison of three AUC techniques for the determination of the loading status and capsid titer of AAVs.
No full textDue to the rise of adeno-associated viruses (AAVs) as gene therapy delivery vectors, boundary sedimentation velocity analytical ultracentrifugation (boundary SV-AUC) has been developed into a widely used quality control assay even for release analytics. It can be considered as the "gold standard" for the determination of the loading status of empty, partially filled, and full capsids especially when conducted in multiwavelength (MWL) mode. It can be considered to provide the most accurate determination of the loading status, and it also provides information on the capsid titer, aggregates, and potential contaminants such as free DNA. MWL boundary SV-AUC can be regarded as a multi-attribute (MAM) method for the characterization of AAVs. One major drawback of the method is the high sample consumption both in terms of concentration and volume. Here, we compare two alternative AUC techniques, band SV-AUC and analytical CsCl density gradient sedimentation equilibrium AUC (CsCl SE-AUC) with the boundary SV-AUC and the MWL-SV-AUC experiment. Our data show a high consistency of the determined full/empty ratios between these techniques if the appropriate wavelengths and extinction coefficients are used
cIAP1/2 antagonism induces antigen-specific T cell dependent immunity
No full textCheckpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naïve T cells can receive co-stimulation not only via CD28 but also through TNF superfamily receptors that signal via NF-B. Antagonists of the ubiquitin ligases cIAP1/2, also called SMAC mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-B signaling that mimics co-stimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Here, we use in vivo models of syngeneic pancreatic cancer that generate endogenous T cell responses ranging from moderate to poor. Across multiple models, cIAP1/2 antagonism has pleiotropic beneficial effects on anti-tumor immunity, including increased activation of dendritic cells and direct effects on tumor-specific T cells leading to overall increased activation, increased control of tumor growth in vivo, synergy with multiple immunotherapy modalities, and immunologic memory. In contrast to checkpoint blockade, cIAP1/2 antagonism does not increase intratumoral T cell frequencies. Furthermore, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent anti-tumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses
FAIR in action - a flexible framework to guide FAIRification.
No full textThe COVID-19 pandemic has highlighted the need for FAIR (Findable, Accessible, Interoperable, and Reusable) data more than any other scientific challenge to date. We developed a flexible, multi-level, domain-agnostic FAIRification framework, providing practical guidance to improve the FAIRness for both existing and future clinical and molecular datasets. We validated the framework in collaboration with several major public-private partnership projects, demonstrating and delivering improvements across all aspects of FAIR and across a variety of datasets and their contexts. We therefore managed to establish the reproducibility and far-reaching applicability of our approach to FAIRification tasks
The ambivalence towards neuropsychology in dementia research, diagnosis, and drug development: Myths and misconceptions.
No full textClinical assessments remain the gold standard for diagnosing neurodegenerative dementia and monitoring disease progression and treatment effects as well research. However, rapid soluble biomarker developments hold promise for increasingly targeted therapeutic approaches, targeted selection of participants in clinical trials, and more direct physiological efficacy readouts. Unfortunately, the anchoring of biomarker research to clinical symptomatology and disease progression is often based on brief and uninformative cognitive tests or screening tools that lack sensitivity to the early stages of cognitive decline. The use of these tools has given the impression that cognitive symptoms occur relatively late in the disease and that disease progression in the early stages of disease is slow. This can hinder advancements in the field and may lead to treatment interventions occurring too late. Poor cognitive test selection has likely been a factor in the failure record of dementia drug development over the last two decades.
A thorough cognitive assessment is a powerful tool in the hands of an expert neuropsychologist and continues to play a key role in the accurate and early diagnosis of neurodegenerative disease. This clinical assessment is very different from the cognitive testing we traditionally see in dementia biomarker research and drug development. Yet the distinction between the two approaches is unclear to many. This paper aims to elucidate some of the myths and misconceptions around cognitive research in dementia and suggests a way forward to facilitate biomarker and drug development through the improved utility of cognitive assessment tools
RNA binding induces an allosteric switch in Cyp33 to repress MLL1-mediated transcription.
No full textMixed-lineage leukemia 1 (MLL1) is a transcription activator of the HOX family, which binds to specific epigenetic marks on histone H3 through its third plant homeodomain (PHD3) domain. Through an unknown mechanism, MLL1 activity is repressed by cyclophilin 33 (Cyp33), which binds to MLL1 PHD3. We determined solution structures of Cyp33 RNA recognition motif (RRM) free, bound to RNA, to MLL1 PHD3, and to both MLL1 and the histone H3 lysine N6-trimethylated. We found that a conserved α helix, amino-terminal to the RRM domain, adopts three different positions facilitating a cascade of binding events. These conformational changes are triggered by Cyp33 RNA binding and ultimately lead to MLL1 release from the histone mark. Together, our mechanistic findings rationalize how Cyp33 binding to MLL1 can switch chromatin to a transcriptional repressive state triggered by RNA binding as a negative feedback loop