UNASSIGNED: Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs.
UNASSIGNED: MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3 M/mL), 1:1 dilution (from 6 M/mL), or 1:2 dilution (from 9 M/mL) with PLA/5% HA, to achieve uniform concentration at 3 M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6 h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6 h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3 M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis.
UNASSIGNED: MSCs can be cryopreserved up to 9 M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.

Per- and polyfluoroalkyl substances (PFAS) are found in many consumer and industrial products. While some PFAS, notably perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are developmentally toxic in mammals, the vast majority of PFAS have not been evaluated for developmental toxicity potential. A concentration-response study of 182 unique PFAS chemicals using the zebrafish medium-throughput, developmental vertebrate toxicity assay was conducted to investigate chemical structural identifiers for toxicity. Embryos were exposed to each PFAS compound (≤100 μM) beginning on the day of fertilization. At 6 days post-fertilization (dpf), two independent observers graded developmental landmarks for each larva (e.g., mortality, hatching, swim bladder inflation, edema, abnormal spine/tail, or craniofacial structure). Thirty percent of the PFAS were developmentally toxic, but there was no enrichment of any OECD structural category. PFOS was developmentally toxic (benchmark concentration [BMC] = 7.48 μM); however, other chemicals were more potent: perfluorooctanesulfonamide (PFOSA), N-methylperfluorooctane sulfonamide (N-MeFOSA), ((perfluorooctyl)ethyl)phosphonic acid, perfluoro-3,6,9-trioxatridecanoic acid, and perfluorohexane sulfonamide. The developmental toxicity profile for these more potent PFAS is largely unexplored in mammals and other species. Based on these zebrafish developmental toxicity results, additional screening may be warranted to understand the toxicity profile of these chemicals in other species.

Stability and potency improvement have been reported by reacting levofloxacin (LF) with citric acid (CA) in a (1:1) molar ratio. However, CA is known to be irritant to the gastrointestinal tract and should be minimized. In a novel approach, this experiment aimed to prepare LF - CA salt with reduced CA, the (2:1) molar ratio, study the structure, and investigate its solubility, stability, and potency improvement. Solvent-dropped grinding and slow evaporation methods were used to prepare the new ratio composition salt, characterized by electrothermal, differential scanning calorimetry, and powder X-ray diffractometry to confirm the physically new solid-state formation. Next, Fourier transform spectrophotometry identified the chemical interaction between LF and CA. After that, a comprehensive structural study using single-crystal X-ray diffractometry determined the 3D structure of the new salt, which determined the solid physicochemical behavior. Finally, stability, solubility, and potency tests were done to investigate the benefits of the new LF-CA composition. As a result, this experiment successfully synthesized the salt, which bound 4.5 water molecules, named LFCA (2:1) - 4.5 hydrate. This new solid-state salt was comparable with the established (1:1) molar ratio in solubility, stability, and potency, higher than LF alone. Hereafter, with a reduced CA portion, this new composition holds potential for further development in drug formulation as a stable, safer, and more efficient antibiotic.

Antimicrobial peptides (AMPs) are recognized for their potential application as new generation antibiotics, however, up to date, they have not been widely commercialized as expected. Although current bioinformatic tools can predict antimicrobial activity based on only amino acid sequences with astounding accuracy, peptide selectivity and potency are not foreseeable. This, in turn, creates a bottleneck not only in the discovery and isolation of promising candidates but, most importantly, in the design and development of novel synthetic peptides. In this paper, we discuss the challenges faced when trying to predict peptide selectivity and potency, based on peptide sequence, structure and relevant biophysical properties such as length, net charge and hydrophobicity. Here, pore-forming alpha-helical antimicrobial peptides family isolated from anurans was used as the case study. Our findings revealed no congruent relationship between the predicted peptide properties and reported microbial assay data, such as minimum inhibitory concentrations against microorganisms and hemolysis. In many instances, the peptides with the best physicochemical properties performed poorly against microbial strains. In some cases, the predicted properties were so similar that differences in activity amongst peptides of the same family could not be projected. Our general conclusion is that antimicrobial peptides of interest must be carefully examined since there is no universal strategy for accurately predicting their behavior.

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRβ) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches.

BACKGROUND: Minimally invasive techniques have demonstrated several advantages over the open approach. In the field of prostate cancer, the LAP-01 trial demonstrated the superiority of robotic-assisted radical prostatectomy (RARP) over laparoscopic radical prostatectomy (LRP) when comparing continence at 3-month after surgery, with no statistically significant differences at 6 and 12 months of follow-up.
OBJECTIVE: Externally validate the LAP-01 study and compare functional outcomes between the two minimally invasive approaches.
METHODS: This retrospective study, conducted by a single surgeon (MRB), utilized data from a prospectively collected database, which included patients who underwent both RARP or LRP. Data regarding baseline characteristics, continence (assessed through the 24-h Pad test and ICIQ questionnaire) and potency were collected at multiple time points: 1 and 6 weeks after catheter removal, 3-, 6-, and 12-months post-surgery.
RESULTS: The study encompasses 601 patients, 455 who underwent LRP and 146 RARP. The median age at diagnosis was 64 for LRP and 62 for RARP, while the median PSA levels at diagnosis were 6.7 ng/mL for LRP and 6.5 ng/mL for RARP. Bilateral nerve-sparing procedures were performed in 34.07 % of LRP cases and 51.37 % of RARP cases. RARP exhibited a significant advantage over LRP both in continence and potency. Continence rates at 3-, 6- and 9-month after radical prostatectomy (RP) were 36.43 %, 61.86 % and 79.87 % for LRP, compared to 50.98 %, 69.87 % and 91.69 % for RARP. Potency rates at the same intervals were 0.90 %, 3.16 % and 6.39 % for LRP, and 6.19 %, 9.16 % and 18.96 % for RARP. These rates were more pronounced in patients with bilateral nerve-sparing.
CONCLUSIONS: Our study demonstrates that RARP results in significantly better continence recovery and superior potency outcomes throughout the entire follow-up period compared to LRP, even at the beginning of the robotic approach learning curve.

During the drug development process, testing potency plays an important role in the quality assessment required for the manufacturing and marketing of biologics. Due to multiple operational and biological factors, higher variability is usually observed in bioassays compared with physicochemical methods. In this paper, we discuss different sources of bioassay variability and how this variability can be statistically estimated. In addition, we propose an algorithm to estimate the variability of reportable results associated with different numbers of runs and their corresponding OOS rates under a given specification. Numerical experiments are conducted on multiple assay formats to elucidate the empirical distribution of bioassay variability.

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

The growing use of botulinum neurotoxins (BoNTs) for medical and aesthetic purposes has led to the development and marketing of an increasing number of BoNT products. Given that BoNTs are biological medications, their characteristics are heavily influenced by their manufacturing methods, leading to unique products with distinct clinical characteristics. The manufacturing and formulation processes for each BoNT are proprietary, including the potency determination of reference standards and other features of the assays used to measure unit potency. As a result of these differences, units of BoNT products are not interchangeable or convertible using dose ratios. The intrinsic, product-level differences among BoNTs are compounded by differences in the injected tissues, which are innervated by different nerve fiber types (e.g., motor, sensory, and/or autonomic nerves) and require unique dosing and injection sites that are particularly evident when treating complex therapeutic and aesthetic conditions. It is also difficult to compare across studies due to inherent differences in patient populations and trial methods, necessitating attention to study details underlying each outcome reported. Ultimately, each BoNT possesses a unique clinical profile for which unit doses and injection paradigms must be determined individually for each indication. This practice will help minimize unexpected adverse events and maximize efficacy, duration, and patient satisfaction. With this approach, BoNT is poised to continue as a unique tool for achieving individual goals for an increasing number of medical and aesthetic indications.

Background: We detail our approach and experience with a hybrid version of the endopelvic hood-sparing (HS) robot-assisted radical prostatectomy (RARP) using the da Vinci robotic platform. Materials and Methods: We retrospectively reviewed the records of 200 patients who underwent RARP by a single surgeon. Patients were propensity-matched into three cohorts depending on biopsy and prostatectomy Gleason Grade Groups: traditional retropubic (RP) (n = 80), retzius-sparing (RS) (n = 40), and HS (n = 80). Patient characteristics and oncologic and functional outcomes were examined. Zero pads per day defined return of continence. Erections suitable for penetrative intercourse with/without medications defined return of sexual function. Results: Patient characteristics were similar between cohorts excluding prostate-specific antigen levels (p = 0.014), which were significantly lower in the RS cohort (7.1 ± 5.3 ng/mL) compared with RP (9.2 ± 9.3 ng/mL) and HS (8.8 ± 8.9 ng/mL). Clinically significant positive margin rates were significantly higher (p = 0.046) in the RS cohort (32.5%) compared with RP (17.5%) and HS (13.9%). Biochemical recurrence and metastasis rates were similar between all cohorts. Median time to continence was significantly lower for RS and HS-RARP (p < 0.001) compared with RP-RARP at 1.3, 1.6, and 5.4 months, respectively. Median time to return of sexual function was significantly lower for RS and HS-RARP (p < 0.001) compared with RP-RARP at 4.0, 7.7, and 15.1 months, respectively. Conclusions: Our hybrid HS-RARP approach provides functional outcomes similar to RS-RARP with the early oncologic control of traditional RP-RARP.