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Technical Considerations of Pharmacokinetic Assays for LNP-mRNA Therapeutics by RT-qPCR
No full textLipid nanoparticle-messenger RNA (LNP-mRNA) therapeutics are a growing class of drug modalities. The unique composition of these therapeutics requires multiple measurements to account for the different components of these drug modalities. Pharmacokinetic (PK) measurements include measurement of the encapsulated mRNA and components of the LNP in circulation to understand the effectiveness of the therapeutic mRNA. The PK measurements can utilize many different platforms including PCR. Current regulatory guidance documents for bioanalytical method validation are specific to ligand binding and chromatographic assay methods and difficult to interpret for use with molecular workflows. The purpose of this paper is to provide information on considerations for validation of regulated reverse transcription quantitative PCR (RT-qPCR) assays that are used to support the pharmacokinetic analysis of LNP-mRNA therapeutics
Evaluation of various experimental conditions and mechanistic static vs. dynamic models to predict time-dependent CYP3A4/5 inhibition potential of drugs
No full textThe use of mechanistic static and dynamic physiologically-based pharmacokinetic (PBPK) models by incorporating cytochrome P450 (CYP)3A4/5-mediated time-dependent inhibition (TDI) parameters from human liver microsomes (HLM) can potentially give rise to significant overprediction of drug-drug interactions (DDI) caused by TDI, which may result in conducting unnecessary clinical DDI trials. This work aimed to evaluate the predictive performance of mechanistic static and dynamic PBPK models employed to predict the likelihood and the magnitude of clinical DDI caused by drugs with in vitro CYP3A4/5 TDI parameters measured in HLM and human hepatocytes (HHEP). We examined the effect of differences in in vitro CYP3A4/5 TDI parameters such as the inhibition constant (KI) (total or unbound) in experimental conditions (supplementation of glutathione in HLM incubations or plasma in HHEP incubations) on the magnitude of predicted DDI risk in comparison to clinical results. In mechanistic static models, the average unbound organ exit concentrations and the maximum organ entry concentrations were compared for projecting DDI risks. Model performance was assessed using false-negative rates and negative predictive errors for a cutoff value of either 1.25- or 2-fold change in midazolam exposure. DDI caused by CYP3A4/5-mediated TDI was reliably predicted using mechanistic static model with average unbound organ exit concentrations or dynamic PBPK modeling, yielding less marked overpredictions of DDI. Models using in vitro KI corrected for incubation unbound fraction generated in either HLM or HHEP buffer incubations showed best statistical performance while maintaining high prediction accuracy and precision
Readiness for Implementation of ICH Q2(R2) and Q14: Results of a Cross-Industry Survey
No full textTo assess industry readiness for the implementation of these guidelines and to understand challenges, opportunities, and overall preparedness of industry professionals, the ISPE-PQLI Analytical Method Strategy (ICH Q2(R2)/Q14) team conducted a comprehensive survey. The survey was conducted anonymously from April 18 to July 15, 2024, receiving 201 recorded responses. Fifty-seven of those responses provided answers to questions beyond the demographic information and are discussed below. These 57 responses are summarized along with optional free text responses provided by the respondents, and the findings and key takeaways from the survey are presented
Transcriptional changes in non-human primate tissues after intrathecal delivery of serotype 9 adeno-associated viral vector: Insights into organ toxicities.
No full textAdeno-associated virus (AAV)-based gene transfer has brought transformative therapeutic benefits to patients with otherwise untreatable genetic diseases. However, treatment-related organ toxicities, particularly for high doses, remain a safety concern in the clinic with some translatability in preclinical species. In the present study, we conducted an RNA sequencing (RNA-seq) analysis in non-human primates administered intrathecally with scAAV9-CBA-GFP, empty viral capsid particles, or a "Promoterless" vector. This analysis revealed a broad and long-lasting (4 weeks after dosing) transcriptional impact of the viral transduction/transgene expression on tissues. Liver and dorsal root ganglia (DRGs), known to be the primary sites of toxicity induced by AAV9, had the highest viral load and the most significant transcriptional changes. Our analysis revealed that most of the differentially expressed genes were upregulated and common gene signatures belonged to immune pathways (innate and adaptive), demonstrating a persistent low-grade immune response up to 4 weeks post-dosing. Interestingly and across all tissues considered, the impact of empty capsids or of the Promoterless vector was minimal, suggesting that the presence of the capsid and a productive viral genome causes the observed changes. This study provides unique insights into the transcriptional responses to AAV9 in key tissues primarily exposed by the vector
Assessment of pharmacokinetic drug interaction of asciminib with atorvastatin in healthy participants
No full textAsciminib is the first BCR::ABL1 inhibitor that Specifically Targets the ABL Myristoyl Pocket (STAMP) in patients with chronic myeloid leukemia. This phase I, two-treatment-period, drug-drug interaction study evaluated the effect of steady-state asciminib on the pharmacokinetics of atorvastatin. A single dose of atorvastatin (20 mg) was administered on day 1 (period 1: days 14). On days 511 (period 2: days 512), 80 mg asciminib was administered once daily, with a single dose of atorvastatin co-administered on day 9. Pharmacokinetic sampling for atorvastatin was performed on days 14 in period 1 and days 912 in period 2. Twenty-two healthy participants were enrolled. Twenty participants completed the study, and two discontinued due to adverse events (AEs). Asciminib increased the adjusted geometric mean (Gmean) of maximum plasma concentration (Cmax), the area under the curve (AUC) from zero to the last quantifiable concentration (AUClast), and the AUC from zero to infinity (AUCinf) of atorvastatin by 24%, 16%, and 14%, respectively, and did not affect these parameters for its active metabolites, o-hydroxy-atorvastatin and p-hydroxy-atorvastatin. Plasma concentrations of coproporphyrin-1 (CP-1), an endogenous substrate of the atorvastatin transporter OATP1B, were not affected by asciminib. Thirteen participants reported at least one AE, all being grade 1/2, except for one grade 3 AE (increased alanine aminotransferase). No serious AEs were reported. In conclusion, concomitant administration of steady-state asciminib and atorvastatin resulted in a small, clinically irrelevant increase in atorvastatin exposure and no change in CP-1 concentrations. Both drugs were well tolerated. These data support co-administration of asciminib and atorvastatin
Development, qualification, and application of a highly efficient and robust new peak detection workflow for the LC-MS peptide mapping multi-attribute method.
No full textThe multi-attribute method (MAM) by liquid chromatography-mass spectrometry peptide mapping has the potential to replace multiple conventional HPLC- and capillary electrophoresis-based purity/impurity assays for release and stability testing of protein biopharmaceuticals such as monoclonal antibodies. Prerequisite is the availability of the new peak detection (NPD) functionality to reliably detect new, absent, and changed peptide species that may impair the quality, safety, and efficacy of the drug. Here, we describe the development, qualification, and application of a highly efficient and robust NPD workflow within the Genedata Expressionist® software. The detection thresholds have been rationally designed, and the NPD workflow has been successfully validated according to ICH Q2 guidelines. Individual case studies, including stability testing of drug product and detection of unknown impurities in drug substance, highlight the workflows' ability to reliably recognize relevant peptide species below 1% relative abundance without reporting any false positive peaks. The application of this NPD workflow signifies a substantial leap forward in the use of MAM as a quality control tool, as it allows identification of true positive peaks at adequate sensitivity in the absence of false positive peaks
Single Cell Foundation Models Evaluation (scFME) for In-Silico Perturbation
No full textFoundation models pre-trained on large single-cell RNA atlas present a com�pelling alternative to in-vitro experimentation for understanding gene regulatory networks and conducting gene perturbation analyses, with significant implica�tions to target identification. Numerous foundation models have been developed, expanding upon early efforts such as Geneformer and scGPT, and they are also configurable through hyper-parameterization, resulting in multiple models and instances that require comparative analysis. Current benchmarking focuses on feature-based assessments or employing intuitive biological and statistical tasks that may not align with the models’ training objectives. A recent study proposed a systematic benchmarking; however, its scope was limited to pre-trained (zero�shot) models. To address these limitations, we propose Single-Cell Foundation Model Evaluation (scFME), a systematic method designed for benchmarking fine-tuned foundation models for in-silico perturbation (ISP). scFME ensures comprehensive and robust assessment by requiring sufficient separation between control and perturbed cells to begin with and quantifying ISP accuracy against zero and random perturbations baselines. Furthermore, scFME enables explo�ration of models’ performance across distinct gene categories enabling biological implications and functional relevance. Using this framework, we assessed several commonly used models (and some of their variants) and demonstrated that the methodology allows us to characterize their performance for ISP studies. Our results position scFME as a versatile and rigorous methodology for the evaluation
and comparison of current and future foundation models
A deep dive into spin-labeled polysorbate's interaction with therapeutic antibody using 2D NMR, EPR and MD simulations.
No full textPolysorbates (PS) are widely used surfactants in biopharmaceutical formulations playing a crucial role in protecting proteins against mechanical stress and interface-induced damage. However, their susceptibility to degradation can compromise their function and lead to particle formation. Recent studies suggest that monoclonal antibodies (mAbs) may mitigate PS degradation catalyzed by histidine chloride buffer, indicating the presence of protein-PS interactions. In this study, we investigated these interactions using NMR, starting with H T CPMG filter experiments and methyl fingerprinting, which failed to detect interactions. To enhance sensitivity, we synthesized spin-labeled PS (SLPS), enabling paramagnetic relaxation enhancement (PRE) NMR experiments, specifically amide fingerprinting, which successfully revealed interactions. Complementary electron paramagnetic resonance (EPR) measurements of SLPS and mAb also detected interactions, but only in the presence of sucrose, underscoring their weak and transient nature. Additionally, molecular dynamics simulations identified potential interaction hotspots on the antibody structure, providing mechanistic insights into these interactions
Additive effects of the new viscosity-reducing and stabilizing excipients for monoclonal antibody formulation.
No full textThe subcutaneous administration of biopharmaceuticals is advantageous over intravenous administration, particularly with regard to improved patient compliance. However, in highly concentrated protein formulations lower viscosity of the formulation and stability of the protein is difficult to achieve. One approach involves using the viscosity-reducing excipients to diminish the interactions between protein molecules. In this context, the main objective of the study was to develop an optimal formulation for a model monoclonal antibody (mAb) and to evaluate new test compounds as viscosity-reducing agents. The test compounds were investigated both individually at increasing concentrations up to 200 mM and in combinations for their viscosity-reducing effect. Our results showed that all individual test compounds reduced the viscosity of the mAb formulation by more than 30 %, with reduction achieved by the six test compounds exceeding that achieved by proline (Pro). A reduction in the viscosity of the formulation below the 20 mPas threshold was achieved either by combining two test compounds or by increasing the concentration of a single compound above 25 mM. An accelerated stability study showed similar stabilization effects regardless of whether the test compounds were used alone or in combination. The percentage of aggregates was below 5 % in most formulations. These viscosity-reducing and stabilization effects corresponded to the dynamic light scattering results, which indicated that the test compounds reduced the attractive forces between the mAb molecules
Development of a safe and efficient continuous flow method for the synthesis of 3-difluoromethoxypyridine derivatives
No full textThe development of a safe, efficient and scalable continuous-flow-chemistry protocol for the O-difluoromethylation of two 3-hydroxypyridine building blocks is described. This example highlights that continuous flow chemistry has become firmly established within Novartis Biomedical Research, and when implemented appropriately can enable the continuous supply of material from the first realization of a potentially interesting intermediate within a discovery project all the way through to clinical evaluation