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Why is Biomarker Assay Validation Different from that of Pharmacokinetic Assays?
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Transmission Electron Microscopy of the Peripheral Nervous System: Methods, Sample Preparation, and Case Studies.
No full textRecommendations and practical considerations for sample collection, fixation, preparation and imaging of peripheral nerves and ganglia for resin embedding, light microscopic evaluation and transmission electron microscopy for ultrastructural pathology evaluation, based on the experience of the authors. Uses and limitations of transmission election microscopy in nonclinical safety studies is demonstrated through case studies of control and test article-dosed nonclinical material to highlight common artifacts and findings in this increasingly important tissue for emerging pharmaceutical modalities
A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome.
No full textThe biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated "dark matter" includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties
Tipping Point Sensitivity Analysis for Missing Data in Time-to-Event Endpoints: Model-Based and Model-Free Approaches
No full textMissing data frequently occurs in clinical trials with time-to-event endpoints, often due to administrative censoring. Other reasons, such as loss-to-follow up and patient withdrawal of consent, can violate the censoring-at-random assumption hence lead to biased estimates of the treatment effect under treatment policy estimand. Numerous methods have been proposed to conduct sensitivity analyses in these situations, one of which is the tipping point analysis. It aims to evaluate the robustness of trial conclusions by varying certain data and/or model aspects while imputing missing data. We provide an overview of the missing data considerations. The main contribution of this paper lies in categorizing and contrasting tipping point methods as two groups, namely model-based and model-free approaches, where the latter is under-emphasized in the literature. We highlight their important differences in terms of assumptions, behaviors and interpretations. Through two case studies and simulations under various scenarios, we provide insight on how different tipping point methods impact the interpretation of trial outcomes. Through these comparisons, we aim to provide a practical guide to conduct tipping point analyses from the choice of methods to ways of conducting clinical plausibility assessment, and ultimately contributing to more robust and reliable interpretation of clinical trial results in the presence of missing data for time-to-event endpoints
Impurity Qualification Requirements for Drug-Linkers Related Impurities Used to Generate Antibody-Drug Conjugates
No full textThis position paper presents a science-based, holistic assessment of the toxicological risks posed by organic impurities in drug-linkers (D-L) used as intermediates in the production of Antibody-Drug Conjugates (ADCs) for oncology indications. The analysis outlined in this manuscript demonstrates that conjugatable organic impurities present at levels at or below 1.0% w/w or 1 mg/day in D-L intermediates are unlikely to result in adverse toxicological effects upon administration of the final ADC drug product. Due to the extremely low exposure (molar and weight) associated with 1.0% w/w impurity level in the DLs, the authors propose to maintain the ICH Q3A criteria of not more than 1 mg/day limit however increase the ICH Q3A qualification limit criteria from 0.15% to 1.0% w/w. Additionally, a methodology is introduced to assess the level of concern for non-conjugatable organic impurities, utilising process-specific and ADC-specific factors to justify specifications for these impurities in D-L intermediates. Based on the minimal risk associated with D-L impurities at or below the discussed levels, the paper proposes a workflow for quality risk management of such impurities in D-Ls
Efficient host cell protein clearance: A study of membrane adsorbers and resins in biopharmaceutical processes.
No full textIn recent years, biopharmaceutical purification processes have shifted towards more productive and cost-effective methods. Membrane technology has emerged as viable alternative to conventional resins, capable of delivering similar product quality while facilitating a simpler and more flexible downstream purification process. This study compares the impurity removal efficiency of three anion-exchange media types (quaternary amine-functionalized agarose resin (Q Sepharose™ Fast Flow), cellulose membrane adsorber (Sartobind® Q), and a hybrid purifier, composed of two complementary anion-exchange media, a quaternary ammonium functional non-woven and a guanidinium functional polyamide membrane (3M™ Polisher ST)) in a flow-through mode during a monoclonal antibody product purification. The content of residual major impurities-host cell proteins (HCPs) and aggregates-were investigated using a design of experiments (DoE) approach, varying pH, ionic strength, and loading densities. Membrane-based devices exhibited high impurity removal capacity, with 3M™ Polisher ST capable of reducing HCP levels from 8000 ppm down to as low as 10 ppm at competitive loading densities in this case study. The presence of critical HCPs, such as esterases capable of hydrolysing ester bonds in polysorbates, was monitored using high-throughput enzyme assays and liquid chromatography-tandem mass spectrometry. Orthogonal HCP analytical methods were required for more informed process development, as esterase presence did not follow the same trend as the general HCP population. All chromatographic media were further tested for their robustness by conducting breakthrough experiments at an optimal pH of 6.5 and conductivity of 4 mS/cm. An additional orthogonal polishing step was required as anion-exchange chromatography alone was insufficient at clearing aggregates. Among options evaluated, 3M™ Polisher ST demonstrated the greatest potential for simplifying the purification process and enhancing productivity
Considerations for Microbiological Control Strategy during Oligonucleotide Drug Substance Manufacturing
No full textEnsuring the quality and safety of synthetic oligonucleotide drug substances demands stringent microbial contamination control. While chemical synthesis inherently carries a lower risk compared to biological manufacturing, robust controls remain critical to minimize potential microbial proliferation at specific stages of the process. Given the limited regulatory guidance directly addressing oligonucleotides, effective contamination control strategies must be built upon thorough risk assessments and established best practices.
This paper, drawing on the collective expertise of the European Pharma Oligonucleotide Consortium (EPOC), provides comprehensive recommendations for microbiological control in oligonucleotide manufacturing. Key points include facility design, environmental monitoring, equipment cleaning, in-process controls, and analytical methods. A thorough risk assessment and a holistic approach to microbial management are advocated. Detailed methodologies for risk evaluation, mitigation, and acceptance of residual risks are outlined. This strategic framework aims to proactively manage potential microbiological hazards, ensuring the consistent production of high-quality oligonucleotide therapeutics
Accurate and reliable detection of clonal hematopoiesis in plasma cell-free DNA.
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Iptacopan for Immune Thrombocytopenia and Cold Agglutinin Disease: A Global Phase II Basket Clinical Trial
No full textIptacopan is a first-in-class, oral, selective inhibitor of complement factor B that has demonstrated positive efficacy across several complement-driven diseases. Here we evaluate the efficacy and safety of iptacopan monotherapy in adult patients with primary immune thrombocytopenia (ITP) and primary cold agglutinin disease (CAD). We performed a global, multicenter, phase II basket study (NCT05086744) enrolling patients with primary ITP or CAD after failure of ≥1 unique prior therapies. Primary endpoints were platelet response (≥50×109/L) for ITP and hemoglobin response (≥1.5 g/dL increase) for CAD sustained for ≥2 consecutive weeks during the first 12 weeks of iptacopan treatment, without the use of rescue therapy. Other endpoints included time to first response, duration of response, pharmacokinetics, safety/tolerability, and FACIT-Fatigue. Nineteen patients were treated with iptacopan (9 ITP, 10 CAD). Five (50%) patients with CAD met the primary endpoint, with a mean increase in hemoglobin from baseline to week 12 of 2.2 g/dL. Improvements were also observed for other outcomes in CAD, including lactate dehydrogenase, bilirubin, reticulocytes, and FACIT-Fatigue. Conversely, no patients with ITP met the primary endpoint. Most treatment-emergent adverse events (TEAEs) were mild, the most common being headache (21%), asthenia (16%), fatigue (16%), and petechiae (16%). In conclusion, iptacopan monotherapy demonstrated encouraging preliminary efficacy in primary CAD, while no protocol-defined responses were observed in primary ITP. Iptacopan may represent a promising oral option for CAD and was well tolerated in both ITP and CAD with no unexpected safety signals
Multi-Modality Investigative Pathology: Leveraging the Full Potential of Spatial Pathobiology in Pharmaceutical Therapeutic Discovery and Development
No full textContemporary pathology is increasingly multimodal and requires the application and integration of newer molecular methods to complement classical morphologic evaluation. In diagnostic medical pathology, especially cancer pathology, integrative diagnoses combining morphology, immunohistochemistry, and molecular data including methylation profiling and clinical sequencing are often routine. Such integrative approaches have refined classifications of diseases and enhanced therapeutic decision making. However, in toxicologic pathology, the integration of multiple modalities is still in its nascency. Many pathologists do not have direct and cost-effective access to the information and expertise needed to leverage the availability of advanced molecular modalities. This half-day continuing education course sought to familiarize participants with the evolution of pathology from its early days to state-of-the art applications of molecular, spatial, and computational pathology, and to encourage them to adopt advanced molecular modalities in their workflows to enhance their morphologic pathology practice