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Reliable and sensitive characterization assays are important determinants of the successful clinical translation of immunotherapies. For the assessment of cytolytic potential, the chromium 51 (51Cr) release assay has long been considered the gold standard for testing effector cells. However, attaining the approvals to access and use radioactive isotopes is becoming increasingly complex, while technical aspects [i.e. sensitivity, short (4-6 hours) assay duration] may lead to suboptimal performance. This has been the case with our ex vivo expanded, polyclonal (CD4+ and CD8+) multivirus-specific T cell (multiVST) lines, which recognize 5 difficult-to-treat viruses [Adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpes virus 6 (HHV6)] and when administered to allogeneic hematopoietic stem cell (HCT) or solid organ transplant (SOT) recipients have been associated with clinical benefit. However, despite mediating potent antiviral effects in vivo, capturing in vitro cytotoxic potential has proven difficult in a traditional 51Cr release assay. Now, in addition to cytotoxicity surrogates, including CD107a and Granzyme B, we report on an alternative, vital dye -based, flow cytometric platform in which superior sensitivity and prolonged effector:target co-culture duration enabled the reliable detection of both CD4- and CD8-mediated in vitro cytolytic activity against viral targets without non-specific effects.

• High variability of in vivo potency assays for wP (pertussis), IPV and menB vaccines. • Low precision makes in vivo assays unsuitable to monitor manufacturing consistency. • Large differences between individual mice likely contribute to menB assay variability. For the batch release of vaccines, potency release assays are required. Non-animal in vitro tests have numerous advantages and are preferred; however, several vaccines are still released using in vivo assays. Their major drawback is the inherent variability with its practical implications. We quantified the variability of in vivo potency release assays for whole-cell pertussis, inactivated polio and meningococcal B (MenB) vaccines which showed large CV (Coefficient of Variation) ranging from 34% to 125%. As inherent variability might potentially be attributed to the highly variable immune system between individual animals, we evaluated the antibody titres to four MenB antigens in 344 individual outbred mice. These varied strongly, with more than 100-fold differences in antibody titres in responsive mice. Furthermore, within individual mice there was generally no correlation between the strengths of the responses to the four antigens. A mouse with a very low or no response to one antigen in many cases exhibited a strong response to another antigen. The large differences between individual animals is likely a considerable contributor to the inherent variability of in vivo potency assays. Our data again support the notion that it is preferred to move away from in vivo potency assays for monitoring batch to batch consistency as part of vaccine batch release testing. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose–response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG–lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines. [ABSTRACT FROM AUTHOR]

Viral diseases represent a major public health concerns and ever-present risks for developing into future pandemics. Antiviral antibody therapeutics, either alone or in combination with other therapies, emerged as valuable preventative and treatment options, including during global emergencies. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well-suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated exceptional efficacy against hematological malignancies, but notably less against solid tumors. To overcome this limitation, it is critical to investigate antitumor CAR-T cell potency in synthetic 3D microenvironments that can simulate the physical barriers presented by solid tumors. The overall goal of this study was the preliminary assessment of a synthetic thermo-responsive material as a substrate for in vitro co-cultures of anti-disialoganglioside (GD2) CAR-T cells and patient-derived glioblastoma (GBM) spheroids. Independent co-culture experiments demonstrated that the encapsulation process did not adversely affect the cell cycle progression of glioma stem cells (GSCs) or CAR-T cells. GSC spheroids grew over time within the terpolymer scaffold, when seeded in the same ratio as the suspension control. Co-cultures of CAR-T cells in suspension with hydrogel-encapsulated GSC spheroids demonstrated that CAR-T cells could migrate through the hydrogel and target the encapsulated GSC spheroids. CAR-T cells killed approximately 80% of encapsulated GSCs, while maintaining effective CD4:CD8 T cell ratios and secreting inflammatory cytokines after interacting with GD2-expressing GSCs. Importantly, the scaffolds also facilitated cell harvesting for downstream cellular analysis. This study demonstrated that a synthetic 3D terpolymer hydrogel can serve as an artificial scaffold to investigate cellular immunotherapeutic potency against solid tumors., (© 2024 Wiley Periodicals LLC.)

Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo. Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, and regeneration.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose–response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG–lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy "Plagencell network" for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <–150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion. [ABSTRACT FROM AUTHOR]

Introduction: In the rapidly evolving landscape of cellular and gene therapy (CGT) products, ensuring potency is paramount for efficacy and patient safety. Decentralized CGT manufacturing requires simplicity to enable analysts with basic training, brevity due to the short shelf life of fresh products, and automation to facilitate efficient reporting with QC/QA management. Here, we present a case study where Orgenesis MD addresses these challenges through the development of a cytotoxicity assay as a potency assay for CAR-T products. Methods: The Axion Biosystems Maestro Z instrument was utilized for real-time monitoring of impedance values within a controlled environment of temperature and CO2. Impedance values served as indicators of target cell coverage and attachment, with decreases indicating target cell death. Target cells were dosed with CAR-T effectors derived from 3 independent donors. Data was processed in the Axion Axis-Z analysis software to obtain impedance values, cytolysis percentage, and dose-response curves. To determine the potency of our CAR-T products we validated specificity, linearity, and accuracy using various batches produced from different viral vectors. The studies identified critical points in the potency assay including defined culture parameters and E:T ratios. The assay demonstrated sensitivity in differentiating between CAR-T batches and revealed notable differences between various vectors used. Unlike conventional endpoint assays, the Axion enables retrospective data analysis and comparison with clinical outcomes, potentially offering valuable insights into CAR-T efficacy over extended co-culture durations. Cytotoxicity assay development addressed critical parameters and demonstrated the sensitivity of the potency assay to discern between the relative potency of CAR-T batches. These findings represent significant progress towards establishing a fully validated potency assay for CAR-T products that addresses the specific needs of decentralized CGT manufacturing. Further qualification and validation studies will refine assay parameters, ensuring robustness across production batches and product constructs, ultimately enhancing the quality and consistency of CGT products and improving patient outcomes. [ABSTRACT FROM AUTHOR]

Background aims. Mesenchymal stromal cells (MSCs) exert broad immunomodulatory functions. We recently demonstrated a strong suppressive effect of MSCs on virus-specific CD8+T-cell proliferation. Here, we further explored the underlying mechanism. Methods. The role of indoleamine 2,3-dioxygenase (IDO) in inhibition of virus-specific CD8+ T-cell proliferation by human MSCs was investigated using a mixed lymphocyte peptide culture assay, IDO intracellular staining and IDO inhibition through 1-methyl-DL-tryptophan (1-MT). Moreover, the influence of the number of passages and the seeding density of MSCs on their IDO expression and immunosuppressive ability were investigated. Results. MSCs with low IDO expression exhibited a reduced suppressive effect on both allo-antigen- and cytomegalovirus (CMV)-specific CD8+T-cell proliferation. 1-MT could partially abrogate the suppressive effect. MSCs inhibited CMV-specific CD8+T-cell proliferation regardless of the number of passages and the seeding density. IDO expression of MSCs was not significantly affected by the number of passages or the seeding density. In addition, the interferon (IFN)-γ level in the culture system was crucial for MSCs to inhibit the proliferation of CMV-specific CD8+ T cells. Summary. MSCs inhibit virus-specific CD8+ T-cell proliferation through IFN-γ-induced IDO activity, resolving current conflicting reports on this issue and indicating the potential need for prophylaxis and surveillance of virus infection in patients treated with MSCs. In addition, our study makes a contribution to the development of MSC potency assay for clinical use. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome.We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection. [ABSTRACT FROM AUTHOR]

A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII ( FVIII) and factor IX ( FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed. [ABSTRACT FROM AUTHOR]

We have developed analytical methods to measure the biological functions of pVGI.1(VEGF2), a naked plasmid DNA product containing vascular endothelial growth factor 2 used in clinical trials for coronary artery diseases (CAD) and peripheral artery diseases (PAD). After being injected into muscles, vascular endothelial growth factor 2 (VEGF-2), presumably expressed in muscle tissues, binds to the endothelial cell receptors VEGFR2 or VEGFR3, triggering the downstream responses including cell proliferation and vascularization. As it is important to make sure clinical material is biological active, we developed a quantitative assay first to measure the receptor binding activity of the pVGI.1(VEGF2) gene product expressed by the transfected host cells, and then a qualitative assay to confirm the cell proliferation promoting activity of the expressed protein. In both assays the signals were plotted directly against input DNA concentrations used to transfect the host cells. We confirmed specificity for both assays. In addition, we demonstrated acceptable levels of spike recovery (86.7-116%), precision (largest relative standard deviation (RSD)=19.3%), linearity and range (60-140% relative potency, 15 - 35 μg/mL) for the quantitative assay. We intend to use the potency assays for routine lot release and stability studies. [ABSTRACT FROM AUTHOR]

Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules. [ABSTRACT FROM AUTHOR]

Potency of pertussis component, present in DPT-vax, is evaluated by Kendrick assay. Strain Bordetella pertussis 18323 is recommended by WHO, which should be stored in a way that ensures its viability, purity and virulence. As part of the assay, the concentration of a strain suspension should be calculated, which opacity is compared at first sight with the Fifth Reference International Preparation of Opacity. This method is very inexact and frequently leads to errors. A seed lot from Bordetella. pertussis 18323 was elaborated and characterized in this study and the concentration of the strain using a calibration curve was calculated. The lot was used in eight potency assays and they complied with established validity criterion. [ABSTRACT FROM AUTHOR]

• Immunoassays without dissolution process was used in antigenicity assessment. • Multiple assays were used for functional characterization of the HEV vaccine. • Feasibility of labelling the vaccine for the WHO CTC use was demonstrated. • No changes in quality attributes of an HEV vaccine at the end of expiry dating. Hepatitis E, which is caused by infection with hepatitis E virus (HEV), is a global health problem in both developed and developing countries. An efficacious hepatitis E vaccine was licensed (by China) in 2011 with a trade name of Hecolin®. The antigen contained in this vaccine is a truncated version of the sole capsid protein encoded by open reading frame 2, which is designated p239. In this study, the real-time and real-condition stability and accelerated stability of five lots of hepatitis E vaccine products at the end of the designated shelf life, were assessed by a well-established quality analysis platform. The protein integrity of p239 that was recovered from the vaccine lots was demonstrated using CE-SDS, LC-MS and MALDI-TOF MS. The particle characteristics of the recovered vaccine antigen were assessed by TEM and HPSEC. The immunogenicity of hepatitis E vaccines was assessed by a mouse potency assay, which is part of product release and stability testing. Several methods were employed to assess the antigenicity of vaccines with or without adjuvant dissolution. Specifically, the well-established methods of sandwich ELISA and surface plasma resonance (SPR)-based BIAcore were used with unique murine monoclonal antibodies. Most interesting, two 'dissolution-free' immunoassays were also used for in situ antigenicity assessment of the vaccines. In addition to the confirmation of vaccine stability at the end of expiry dating, i.e., after storage in recommended conditions (2–8 °C) for 36 months, the mouse potency assay and sandwich ELISA were used to assess the accelerated stability of prefilled syringes to demonstrate the feasibility of out-of-cold-chain storage. In summary, molecular and functional characterization confirmed the shelf life stability of the vaccine at the end of expiry dating and the feasibility of transporting the hepatitis E vaccine for a given period of time out of cold chains. [ABSTRACT FROM AUTHOR]

In vitro retinal organoid modeling from human pluripotent stem cells is becoming more common place in many ophthalmic laboratories worldwide. These organoids mimic human retinogenesis through formation of organized layered retinal structures that display markers for typical retinal cell types. Pivotally these humanized retinal models provide a stepping stone to the clinic as therapeutic tools and are expected to provide a promising alternative to current animal models. Thus pluripotent stem cell based healthy as well as diseased human retinal organoids are attractive for use in drug potency assays and gene augmentation therapeutics. Here we outline an established protocol for generation of these retinal organoids and how they can be used in conjunction with adeno-associated virus vectors for transgene expression assays.

This protocol details on a screening method for infectivity and tropism of different serotypes of adeno-associated viruses (AAVs) on human retinal explants with cell-type specific or ubiquitous green fluorescent protein (GFP) expression vectors. Eyes from deceased adult human donors are enucleated and the retinas are isolated. Each retina is punched into eight to ten 6-mm equal pieces. Whatman™ paper punches are placed on the retinas and the stack is transferred onto 24-well culture inserts with the photoreceptors facing the membrane. AAVs are applied on the retinal explant punches to allow transduction for 48 h. Retinas are nourished by a serum-free Neurobasal®-A based medium composition that allows extended culturing of explants containing photoreceptor inner and outer segments. The protocols include quality control measurements and histological staining for retina cells. The cost and time effective procedure permits AAV transgene expression assays, RNAi knockdown, and pharmacological intervention on human retinas for 21 days ex vivo.

The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.

Comparison of the immunogenicity response and resistance to challenge in the modified intracerebral challenge assay induced by various acellular pertussis vaccines showed that these were not closely linked. The immunogenicity assay was effective for confirming the presence of specific antigenic components and was invaluable for detecting minor components present in co-purified vaccines. However, the magnitude of antibody responses was not consistently related to antigen concentration nor did it correlate with protection in the modified intracerebral challenge assay. The immunogenicity assay detected degradation of pertussis toxin and pertactin components but not of filamentous haemagglutinin or fimbriae 2 and 3 in denatured acellular pertussis vaccines. The modified intracerebral challenge assay was effective in detecting antigen degradation in all types of acellular pertussis vaccines including those of European/North American origin but was dominated by the response to pertussis toxin. Aerosol challenge was more sensitive in detecting denaturation of filamentous haemagglutinin or fimbriae. The modified intracerebral challenge assay was the only assay that provided a quantitative indication of protective activity. Both immunogenicity and challenge assays provided useful data on acellular pertussis vaccine properties but were complementary and not alternatives.