AAPS Journal最新文献

Drug tolerance (DT) is a critical attribute of anti-drug antibody (ADA) assays for assessing clinical immunogenicity. We present a unique situation where a previously approved commercial product, atezolizumab, required re-assessment of the assay DT to meet an increased drug exposure demand arising from a new route of administration (subcutaneous) and align with updated health authority (HA) regulations. Rather than redevelop the existing ADA assay, which could disrupt ongoing clinical trials, we identified a new anti-idiotype (anti-ID) antibody surrogate that demonstrated that the assay maintained adequate DT for the new route of administration. This streamlined approach addressed concerns regarding higher serum trough concentrations with subcutaneous administration and stricter sensitivity expectations. We established a target DT concentration based on population pharmacokinetic modeling to ensure adequate ADA characterization at steady state. This case study highlights the value of having alternative surrogate ADAs and demonstrates that achieving stringent DT requirements can be accomplished without extensive method redevelopment. We also introduce the broader implications of surrogate ADA selection, binding kinetics, and the clinical relevance of achieving high DT in the context of atezolizumab's efficacy and safety profile. This work also emphasizes the importance of considering bioanalytical assay characteristics, such as DT, throughout a product's lifecycle.

To investigate the feasibility of a singlicate-based approach for immunogenicity assays in the biosimilar setting by comparing singlicate and duplicate data of an established Anti-Drug Antibodies (ADA) assay and from a biosimilar study. We re-calculated the screening, confirmatory, and titer cut-points using singlicate values. The ADA method validation initially performed in duplicates was re-evaluated based on singlicate data. We performed variance component analysis to investigate the contribution of well-well variance on overall data variability. We re-assessed the clinical immunogenicity study data based on singlicate values. The ADA assay validation parameters were comparable between duplicate and singlicate-based evaluation. The variance component analysis confirmed the negligible influence of well-to-well variability. The use of singlicates would not have impacted the immunogenicity outcome of the clinical study. The singlicate-based analysis in our study would have reduced the analytical workload by about ~ 40%.

The development of multi-specific biotherapeutics has revolutionized targeted therapy by simultaneously engaging multiple receptors or pathways, thereby enhancing therapeutic efficacy and specificity. However, evaluating the immunogenic potential of these complex molecules remains a significant challenge, particularly in the reliable detection of neutralizing antibodies (NAbs). To support the development of a bispecific biotherapeutic, we initially established a cell-based bioassay utilizing a cytotoxicity assay platform for NAb assessment. This traditional approach faced significant limitations due to severely limited drug tolerance which prevented accurate NAb classification. To address this hurdle, we developed a non-cell based competitive ligand binding (CLB) assay. The primary obstacle was the multi-transmembrane receptor target, which lacks a soluble form suitable for conventional immunoassays. We successfully addressed this challenge by leveraging a novel synthetic version of the multi-transmembrane receptor as the surrogate target. This enabled the development of a duplex competitive ligand binding assay utilizing a homogeneous bead-based AlphaLISA™ assay platform. This innovative duplex NAb assay significantly increased drug tolerance by at least 170-fold when compared to the cell-based assay, enabling sensitive and specific detection of NAb against each drug target binding domain. The AlphaLISA™ based CLB NAb exhibited negligible matrix interference and generated excellent intra-assay and inter-assay precision, with data concordant across different reagent lots and plate readers. Our results demonstrate that the AlphaLISA™ assay platform offers a robust, sensitive, and drug-tolerant alternative to traditional cell-based NAb assays. This approach provides a superior solution for assessment of NAb against multi-specific biotherapeutics targeting multi-transmembrane receptors.

Antibody-Dependent Cellular Cytotoxicity (ADCC) is a key mechanism of action for humoral immune response, which is important for clinical antibodies such as trastuzumab and cetuximab. The level of ADCC is dependent on multiple properties such as antibody isotype, Fab affinity, epitope, and geometry in the immune synapse. Here, we integrated computational simulations with experiments to analyze the impacts of several key factors on ADCC, including binding affinity, target expression, hinge flexibility, and antibody valency. The kinetic model was adapted to simulate antibody cross-linking between tumor and immune-reporter cells, followed by signal activation. Given the complexity of the interactions between cells and the formation of the immunological synapse, we fitted the effective on-rates within the synapse that are hard to determine a priori. With minimal fitting, the model successfully replicated the trends of immune activation for a series of trastuzumab structural mutants. The simulations demonstrated that antibody variants with a higher likelihood of monovalent target binding, such as single-arm antibodies, as well as those with low Fab affinity and reduced hinge flexibility, increased signaling. The model was able to capture the efficacy of mixtures of antibodies with different Fc domains, which are relevant for combination treatments such as trastuzumab and pertuzumab. Interestingly, the fraction of receptors blocked with antibody combinations was more important than total receptor expression, implying restrictions on diffusion of free receptors in the synapse. Overall, the simulations showed close agreement with experimental observations, providing a tool to interpret the ADCC results and guide the design of antibody therapeutics.

Adeno-associated virus (AAV) vectors are made up of a protein shell that typically encapsidates a single stranded viral deoxyribonucleic acid (DNA) genome up to a total of ~ 4700 nucleotides. Classical AAV Manufacturing processes yield a mixture of capsids that either contain no payload, partial DNA, or the gene of interest (GOI). Several studies have generated variable data indicating that empty capsids (1) affect transduction efficiency by engaging the immune system's neutralizing antibodies, (2) do not improve full capsid uptake, (3) cause liver toxicity at high capsid loads, or (4) alter biodistribution. To study the effect of different empty capsid ratios on transduction efficiency, multiple AAV serotypes and full-to-empty methods were utilized in conjunction with a 5-day in vitro potency method. Total protein expression levels for each AAV were measured by a luciferase reporter after differentiation of C2C12 mouse myoblast cells into myotubes. Empty capsid load regardless of serotype identity did yield a statistically significant change in total protein expression levels. This data suggests that limiting the number of empty capsids regardless of the serotype is advised to ensure the highest dose of GOI containing AAVs reach target cells. Manufacturing practices should continue to reduce the overall empty capsid load in each batch to ensure most AAV particles contain the GOI. The findings of this project could aid in process optimization and method development strategies for cell-based assays to quantify the level of protein expression in AAVs containing other GOIs.

Wound healing is a complex process often impaired in severe injuries, requiring innovative therapeutic strategies. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) modulate key cellular pathways, but their clinical application is limited by low stability and bioavailability. This study aimed to evaluate the safety and potential of sodium alginate hydrogels (SAH-EVs) loaded with mesenchymal stem cell-derived extracellular vesicles, focusing on cell migration, cytotoxicity, genotoxicity, and irritation potential. MSC-EVs from Sprague-Dawley rat bone marrow were isolated from conditioned medium collected at 24, 36, 48 and 60 h using size exclusion chromatography and characterized by Nanoparticle Tracking Analysis. The highest EV concentration was obtained from the conditioned medium collected at 36 h, with a main peak at 123 nm. The heterogeneous particle population suggests the presence of EV subtypes. Scanning Electron Microscopy confirmed successful MSC-EVs incorporation into hydrogels with desirable viscoelastic properties. SAH-EVs stimulated HaCaT keratinocyte migration while exhibiting low cytotoxicity in 2D and 3D models, with no genotoxic or mutagenic effects. HET-CAM assays confirmed the absence of irritation potential. These findings highlight the potential of SAH-EVs as a safe biomaterial and lay the groundwork for further investigations into their role in wound healing, reinforcing their relevance in regenerative medicine and tissue engineering.

This study developed a novel aprepitant (APT) nanocrystal-containing oral disintegrating tablet (ODT) to enhance the oral bioavailability of APT through "rapid disintegration". APT nanocrystal suspension (APT-NS) was prepared via miniaturized media milling, with a particle size of 199.25 ± 12.88 nm, a polydispersity index (PDI) of 0.148 ± 0.026 and zeta potential of -24.82 ± 1.28 mV. The APT-NS was spray-dried into dry powders (APT-NCS) and further processed into ODTs via freeze-drying. Through central composite design-response surface methodology (CCD-RSM) optimization, the final APT-ODT formulation demonstrated rapid disintegration (< 5 s) and excellent dissolution (> 95% within 2 min). Results of X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) confirmed the absence of crystalline transformation or chemical degradation during processing. Pharmacokinetic results demonstrated that both APT-NS and APT-ODTs exhibited approximately twofold higher AUC_0-48 h (28.51 ± 1.62 μg·h/mL and 32.61 ± 2.19 μg·h/mL, respectively) compared to free APT. In conclusion, the optimized APT-ODT successfully improved the oral bioavailability of APT, representing a promising new strategy for clinical application.

Antibody-drug conjugates (ADCs) are composed of a tumor-targeting mAb conjugated to a cytotoxic payload to enable the selective delivery of the cytotoxic moiety while reducing the side effects and immunogenicity to the patient. Since the first ADC approval by the FDA, the design of new generation of ADC products has been extensively developed to improve the therapeutic efficiency of first-generation ADCs. Concomitantly, different analytical methods have been improved to enable critical quality attributes (CQA) assessment and thus support ADCs development and production at different stages. In this context, liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) have played a predominant role in ADCs characterization, showcasing the advantages of these methods to identify and potentially quantify the different ADCs populations resulting from the bioconjugation process. This review provides a detailed overview about cutting-edge analytical methods with a particular focus on studies reported during the last five years related to LC, CE, and MS, allowing not only a deeper insight into ADCs structure, but also to provide further evidence about their in vitro and in vivo biotransformation products. The maturity, robustness, and high throughput associated to these methods allow their progressive introduction in regulatory and clinical environments, ensuring the structural integrity of ADCs prior to their administration to the patients. The examples reported in this review article clearly highlight the relevance of using tailored analytical strategies for a more comprehensive ADC characterization and thus continue the contribution to early developability assessment and bring next-generation ADCs into the market.

Target-mediated drug disposition (TMDD) refers to non-linear pharmacokinetic (PK) profiles arising from the saturable interaction between a drug and its pharmacological target. Recently, our group revisited the TMDD cases observed in small-molecule drugs interacting with high-specificity targets, obtaining quantitative insights into in vivo target binding. In this study, we developed a physiologically-based PK (PBPK) model incorporating TMDD and pharmacodynamic (PD) responses (TMDD-PD) for finasteride and dutasteride, two time-dependent inhibitors of 5α-reductase (5αR). In addition to the tissue- and subtype-dependent 5αR inhibition, the model incorporated irreversible inactivation of 5αR and its turnover to account for the mechanism of time-dependent inhibition. We simultaneously analyzed the non-linear PK and PD (dihydrotestosterone level decline and recovery) data for both finasteride and dutasteride. Our model effectively captured the observed PK/PD profiles of both drugs, and the model-derived 5αR inhibition parameters were comparable to those obtained from in vitro 5αR inhibition data. Sensitivity analysis revealed that saturation of target binding is the primary driver of the non-linear PK and corresponding PD profiles, while slow turnover of 5αR contributes to the prolonged PD effect. Our results further suggest that the distinct PD profiles of finasteride and dutasteride are attributable to their differing inhibition characteristics against 5αR subtypes (selectivity and affinity). These findings extend our previous work and further support the utility of TMDD-PD modeling for optimizing clinical dose and improving therapeutic outcomes for small-molecule drugs exhibiting TMDD with time-dependent target inhibition.