Multiparametric grading of glaucoma severity by histopathology can enable post-mortem substratification of disease state.

Neurodegeneration in glaucoma patients is clinically identified through longitudinal assessment of structure-function changes, including intraocular pressure, cup-to-disc ratios from fundus images, and optical coherence tomography imaging of the retinal nerve fiber layer. Use of human post-mortem ocular tissue for basic research is rising in the glaucoma field, yet there are challenges in assessing disease stage and severity, since tissue donations with informed consent are often unaccompanied by detailed pre-mortem clinical information. Further, the interpretation of disease severity based solely on anatomical and morphological assessments by histology can be affected by differences in death-to-preservation time and tissue processing. These are difficult confounders that cannot be easily controlled. As pathogenesis and molecular mechanisms can vary depending on the stage and severity of glaucoma, there is a need for the field to maximize use of donated tissue to better understand the molecular mechanisms of glaucoma and develop new therapeutic hypotheses. Further, there is a lack of consensus around the molecular RNA and protein markers that can be used to classify glaucoma severity. Here, we describe a multiparametric grading system that combines structural measurements of the retinal nerve fiber layer with linear regression and principal component analyses of molecular markers of retinal ganglion cells and glia (RBPMS, NEFL, IBA1 and GFAP) to stratify post-mortem glaucoma eyes by the severity of disease. Our findings show that a quantitative grading approach can stratify post-mortem glaucoma samples with minimal clinical histories into at least three severity groups and suggest that this type of approach may be useful for researchers aiming to maximize insights derived from eye bank donor tissue

Assessing the Adjuvant Potential of Chinese Hamster Ovary Host Cell Proteins Using an In Vitro Dendritic Cell Assay.

Host cell proteins (HCPs) are process-related impurities of therapeutic protein production and may affect product quality or patient safety. In clinical trials, certain HCPs (e.g., PLBL2 or CCL2) that co-purify with the therapeutic protein have been associated with immune reactions in patients. In this study, we examined the adjuvant potential of six commonly detected HCPs from CHO cells (PRDX1, S100A4, PLBL2, CCL2, CLU, and YWHAE) using an in vitro dendritic cell (DC) maturation assay. Recombinant HCPs were expressed in CHO cells to mimic manufacturing conditions. PRDX1, S100A4, and PLBL2 caused a slight increase in the expression of maturation markers on DCs, while YWHAE, CLU, and CCL2 did not. Interestingly, CLU and CCL2 reduced the DC maturation induced by rituximab. In addition, we observed that process parameters such as elution conditions during chromatographic purification can influence HCP aggregation, which in turn can mask or enhance the intrinsic adjuvant potential of an HCP. These findings not only provide initial insights into the adjuvant potential of individual HCPs but also indicate that the quantity as well as the degree of aggregation of HCPs might influence adjuvanticity

Revealing Unpredicted Aspartic Acid Isomerization Hotspots by Probing Diagnostic Fragmentation Propensities in Top-Down and Middle-Down Mass Spectrometry.

Isomerization of aspartic acid residues is a relevant degradation pathway of protein biopharmaceuticals as it can impair their biological activity. However, the in silico prediction of isomerization hotspots and their consequences remains ambiguous and misleading. We have previously shown that all ion differential analysis (AiDA) of middle-down spectra can be used to reveal diagnostic terminal and internal fragments with more sensitivity than the conventional fragment ion mass matching methodology. In this study, we use AiDA to characterize the degradation of an antibody fragment at three aspartic acid isomerization sites including a novel DW motif directly with electron-transfer/higher-energy collisional dissociation top-down and middle-down mass spectrometry. We show that AiDA methodology is pivotal to probe diagnostic fragmentation propensities of terminal c and z fragments at the N-terminus and vicinity of isomerization sites in addition to the diagnostic c+57 terminal fragments. Furthermore, AiDA can probe remote structural changes in the loop of an antibody complementarity-determining region induced by isomerization and the succinimide intermediate, revealing interactions between residues in agreement with molecular simulations. This study shows that aspartic acid residues at noncanonical DW and DF motifs can be hotspots for isomerization despite being ranked as false positives in physics-based prediction models. We show that the enzyme-free, fast, and sensitive AiDA methodology can be used as an orthogonal technique to fractionation for online variant characterization

Scientific and Regulatory Policy Committee Points to Consider: Proposal and Recommendations to Reduce Euthanasia of Control Nonhuman Primates in Nonclinical Toxicity Studies

Nonhuman primates (NHPs) have been and remain a highly valuable animal model with an essential role in translational research and pharmaceutical drug development. Based on current regulatory guidelines, the nonclinical safety of novel therapeutics should be evaluated in relevant nonclinical species, which commonly includes the NHP for biotherapeutics. Given the practical and ethical limitations on availability and/or use of NHPs and in line with the widely accepted guiding “3 Rs” principles, many approaches have been considered to optimize toxicity study designs to meaningfully reduce the use of NHPs. Standard general toxicity studies usually include four groups of equal size, including one group of vehicle control animals. Here we describe an approach to achieve an overall significant reduction in control animal use, while also resolving many of the issues that may limit application of fully virtual control animals. We propose in GLP-compliant safety studies to maintain concurrent control group animals for the in-life phase of the studies, but to limit euthanasia to a subset of control animals. The non-terminated control animals can then be returned to the facility colony for reuse in subsequent studies. The proposed study design could lead to a 15 to 20% reduction in NHP usage. The scientific, logistical, and animal welfare considerations associated with such an approach and suggested solutions are discussed in detail

Single cell multi-omics reveals re-dosing with CD3 bispecific antibody induces a TCF7 high central memory CD8 + T cell population associated with reduced cytokine production.

T cell engaging therapies are commonly accompanied by excessive cytokine production and risk of cytokine release syndrome (CRS). Intriguingly, CRS risk with CD3-engaging bispecific antibody (BSP) is primarily limited to the first dose, termed the first-dose effect. Mechanisms underlying this effect remain unknown. CD3 bispecific induces cytokine cascade via T cell triggering and bystander cells. We hypothesize that distinct T cell biology between doses drives the first-dose effect.We used the Re-directed T Cell Cytotoxicity (RTCC) assay to assess tumor killing and cytokine production by human donor T cells after initial versus subsequent CD3/CD20 BSP treatment. After confirming the first-dose effect in the experimental system containing only T cells and target tumor cells, we employed 10x Genomics single cell multi-omics to study the molecular mechanisms.Compared with initial CD3/CD20 BSP treatment, subsequent treatment exhibited lower cytokine levels and comparable tumor killing. Single cell multi-omics unveiled distinct T cell biology. In initial treatment, T effector memory (Tem) cells are the primary cells that respond to CD3 bispecific antibody stimulus by producing moderate levels of cytolytic and high levels of cytokine gene transcription. In the subsequent treatment, a new population of high TCF7 expressing central memory CD8 + cells (CD8-Tcm-TCF7), possibly originated from stimulated naive T cells, are the primary responding cells that produce a shifted balance with high level of the cytolytic gene transcription (GZMB) and low level of cytokine gene transcription (TNF-alpha and IFN-gamma). Dasatinib co-treatment during initial treatment eliminated cytolytic activity and cytokine production, allowing uncompromised tumor killing and reduced cytokine production upon re-challenge.The distinct T cell populations that respond to first and subsequent CD3 bispecific treatment offer an explanation to the first-dose effect, wherein the risk of CRS associated with CD3 bispecific treatment is mainly limited to the initial dose. Furthermore, our work suggests that tumor killing capacity and cytokine production of T cells could be uncoupled, as demonstrated here by utilizing different T cell populations as effector cells. These findings could be further explored for designing mechanism-based strategies to mitigate the risk of CRS

Do Patient Needs Define Biologic Product Specifications?

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A systemically delivered AAV-CFTR gene therapy approach for cystic fibrosis

Cystic fibrosis (CF) is the most common monogenic lung disease and results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). There have been over 2000 variants identified in patients that can result in loss of function of the CFTR protein leading to systemic disease and respiratory failure in adolescence. While some variants encode proteins with residual activity that can be corrected or potentiated by CFTR modulators, at least 10% of CF individuals cannot tolerate the modulators or have nonsense mutations which fail to make any protein. For all people with CF, a mutation agnostic gene replacement strategy could provide a cure for CF lung disease. Here, we propose using a systemic route of administration to deliver a functional CFTR minigene cargo with a lung tropic AAV capsid. This would serve to reach multiple organs, most importantly the lung epithelium, and would provide a functional CFTR transgene that could be expressed in any cell type with a ubiquitous promoter. To achieve this, we generated the smallest CFTR minigene tested in an AAV delivery to date. We demonstrate it is expressed and functions following transfection in cell-based assays and restores function to primary CF airway cells after viral delivery. Furthermore, we identify an AAV capsid that can transduce alveolar and airway epithelium with systemic delivery in non-human primates. These data provide tools for delivering a functional CFTR minigene that fits within the packaging capacity of an AAV and demonstration of lung transduction following systemic delivery in a large animal model. This strategy would serve to reach target airway cells while circumventing the strong mucosal barrier in CF airways and has the potential to restore CFTR function in additional CF affected organs

Taking Backgrounded Membrane Imaging (BMI) for particle analysis in biopharmaceutics to the next level - Statistical variability, detection limits and novel metrics.

The aggregation of proteins is a major threat to the integrity of biopharmaceutical products. Typically the state of aggregation at a specific timepoint is evaluated via particle analysis and counting or turbidity. Backgrounded Membrane Imaging (BMI) is a recently introduced methodology that provides a low-volume, high-throughput alternative to be used in biopharmaceutical development. Recent work has successfully evaluated BMI as an orthogonal method regarding its counting and sizing accuracy for subvisible particle analysis. The work at hand shows that apart from background noise, stochastic variations need to be considered to define the lower limit of detection. A systematic evaluation of particle identification robustness shows that particles at the lower and upper size limit of the technique are not reliably detected. To overcome potential biases due to particle crowding and overlapping, novel evaluation parameters are introduced: the Total Area, the Total Intensity and the BMI-Z-Average to be reported alongside the particle count. Overall, we were able to refine root causes for loss in data quality in BMI and to showcase the use of additional reporting parameters to shift focus to more robustly-identified and quantified larger particles

Strategies for the evaluation and characterization of higher-order structures, supramolecular higher-order structures, and aggregates in oligonucleotide therapeutics.

Synthetic oligonucleotides have emerged as a promising therapeutic class, holding significant potential for the treatment of a wide range of indications, including previously undruggable targets. Due to specific primary structure motifs, oligonucleotide therapeutics may form higher-order structures (HOS), supramolecular higher-order structures (sHOS) and/or aggregates, which are influenced by factors such as dissolution medium and oligonucleotide concentration. Although rarely observed, unintended (s)HOS and/or aggregates might influence various stages of drug substance (DS) or drug product (DP) manufacturing. There is no published guidance on how to best approach the characterization of oligonucleotide therapeutics (s)HOS and aggregation in a scientific and systematic manner. In this article, we provide an overview of oligonucleotide therapeutics (s)HOS formation and aggregation behavior. Based on industry experience and available literature, we also propose strategies for their evaluation and characterization under relevant conditions. The recommended approach involves conducting appropriate scientific assessments during product development with the support of designed workflows, which can help anticipate and/or mitigate the formation of unintended species

Structural changes of cinnarizine-stabilizer core-shell nano- and micro-suspensions following freeze- and spray-drying determined from dynamic nuclear polarization enhanced NMR.

Developing potent drug molecules that are also highly soluble in aqueous media puts strong constraints on molecular design. As a result, there is intense interest today in developing drug formulation strategies that increase solubility. Specifically, nanosizing involves the reduction of the particle size of the active pharmaceutical ingredient (API) to the sub-micron range, which increases the surface area and dissolution rate. This strategy requires the addition of stabilizers, generally selected through extensive experimental screening, to maintain the desired physical properties over time. To better understand stabilization mechanisms and to develop better future formulations, atomic-level characterization of the particle structures is required in terms of both the size and spatial distribution of the components and the interactions. However, methods that can simultaneously provide this information are scarce. Here, using cinnarizine as a model for nanosuspensions, we show that by using DNP-enhanced NMR we can (i) detect and assign the API and the stabilizers present in formulations; (ii) observe atomic-level API-stabilizer interactions at natural isotopic abundance using two-dimensional 1H-13C correlation NMR experiments; and (iii) determine the domain sizes and the hierarchical structure of the API-stabilizer particles on the nano-meter length scale, based on polarization build-up curves and steady-state enhancements. We then use this approach to evaluate how freeze-drying and spray drying processes, generally used to isolate the material in the solid state, impact the particle structure. More broadly, the results confirm the applicability of DNP-enhanced NMR methods to characterize pharmaceutical suspensions or slurries, and to follow changes upon further processing