Akiko Ishii-Watabe

Therapeutic extracellular vesicle (EV) products, including those containing exosomes (hereinafter referred to as EV products), are expected to have potential as a new therapeutic modality. Many EV products are currently under development in Japan or other countries; however, there are no guidelines specific to this product category and there is a need for establishing regulatory requirements that supports product development. EVs have an extremely complex structure and show high levels of heterogeneity, and their mechanisms of action remain unclear. Additionally, because therapeutic EV products are often manufactured using cells, and it is difficult to sufficiently remove product- or process- related impurities during the purification process, thus posing challenges for impurity control. To ensure the quality of EV products, it is necessary to conduct sufficient characterization of EV products and to identify critical quality attributes that require control to ensure efficacy and safety. Thus, establishing a strategy for controlling the critical quality attributes within the intended limits, ranges, and distributions is key. Furthermore, it is important to set specifications to confirm that EV products of the intended quality have been obtained. This article describes the current state of research on establishing characterization methods for EV products, and recent topics related to quality issues that need to be considered for the development of therapeutic EV products.

Background Promoting the use of biosimilars is a global issue from the perspective of reducing medical costs. In Japan, the replacement of original biopharmaceuticals with biosimilars has not progressed as expected. To promote the use of biosimilar monoclonal antibodies, it is necessary to increase the public’s understanding of biosimilars by evaluating and confirming the quality of biosimilar products. However, there are limited data to compare among multiple biosimilars and/or the reference, and among their product lots. Objective In this study, we evaluated quality attributes of multiple lots of reference products and their biosimilars to determine the extent of distribution in quality attributes between products as well as the quality consistencies from lot to lot. Methods As the quality attributes, the glycosylation profile, charge heterogeneity, binding affinity for the antigen and Fcγ receptors, and high-molecular-weight species and subvisible particles were measured. Results The degree of similarity in quality attributes with a reference product was different for each biosimilar product. We confirmed the differences between reference and biosimilar product reported in previous articles or review reports, and that some quality attributes of biosimilars were out of the quality ranges of reference products. Although lot-to-lot variations and trends at some degrees were observed for some products, no clear drifts among lots were observed. Conclusion Analyzing several lots of these products enabled us to capture a profile of quality characteristics for each product. Overall, the extent of variability of each quality attribute among reference products and biosimilars was revealed by this study for the first time. Graphical Abstract

Background: Extracellular vesicles (EVs), including exosomes, are promising pharmaceutical modalities. They are purified from cell culture supernatant; however, the preparation may contain EVs with the desired therapeutic effects and different types of EVs, lipoproteins, and soluble proteins. Evaluating the composition of particulate impurities and the levels of protein impurities in final preparations is critical for quality control. However, few analytical methods can detect these impurities. Methods: We established and evaluated an analytical method using size-exclusion chromatography–multi-angle light scattering (SEC-MALS) for particle and protein impurity analyses of EV samples. Results: In the particle size distribution analysis of EV samples, SEC-MALS showed higher resolution compared with nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). MALS showed comparable accuracy and precision to that of other methods for particle size evaluation using polystyrene standard beads with 60, 100, or 200 nm diameter. Coupling SEC-MALS with UV detection quantitatively evaluated soluble protein impurities. Proteomic analysis on the SEC-MALS-fractionated samples identified different EV and lipoprotein marker proteins in different fractions. Conclusions: SEC-MALS can characterize EV preparations obtained from human adipose-derived mesenchymal stem cells, suggesting that it can evaluate the particle component composition in various EV samples and therapeutic exosome preparations.

Biopharmaceuticals have developed rapidly in recent years due to the remarkable progress in gene recombination and cell culture technologies. Since the basic structure of biopharmaceuticals can be designed and modified, it is possible to control the duration of action and target specific tissues and cells by kinetic modification. Amino acid sequence modifications, albumin fusion proteins, polyethylene glycol (PEG) modifications, and fatty acid modifications have been utilized to modify the duration of action control and targeting. This review first describes the position of biopharmaceuticals, and then the kinetics (absorption, distribution, metabolism, elimination, and pharmacokinetics) of classical biopharmaceuticals and methods of drug quantification. The kinetic innovations of biopharmaceuticals are outlined, including insulin analog, antibody-related drugs (monoclonal antibodies, Fab analogs, Fc analogs, Fab-PEG conjugated proteins, antibody-drug conjugates, etc.), blood coagulation factors, interferons, and other related drugs. We hope that this review will be of use to many researchers interested in pharmaceuticals derived from biological components, and that it aids in their knowledge of the latest developments in this field.

Monitoring serum infliximab (INF) concentrations is crucial for designing appropriate doses for patients with rheumatoid arthritis. It is recommended to maintain the serum trough INF level at least 1.0 µg/mL. In Japan, an in vitro diagnostic kit using immunochromatography has been approved to determine whether the serum INF concentration is over 1.0 µg/mL or not, and to support the determination of the necessity of increasing the dose or switching to another drug. Biosimilars (BS) of INF may have immunochemical properties different from those of its innovator product, which may show different reactivities on the diagnostic kit. In this study, the responses of the innovator and five BS products on the kit were compared. Based on visually comparing the intensity of color development between the test and control samples, differences were found in the judgment results depending on the analyst. In particular, 1.0 µg/mL was not determined as positive in some cases, whereas 2.0 µg/mL was reliably determined as positive. Overall, no significant difference in reactivity was found between the innovator and five BS products. To further compare the differences in immunochemical properties, the reactivity of these products with three enzyme-linked immunosorbent assay (ELISA) kits was compared. The results confirmed that there were no significant differences among the innovator and BS products in reactivity with the examined kits. When using that diagnostic kit, the users need to be aware that the judgement around 1.0 µg/mL INF may differ depending on the test conditions, including the analyst.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)–specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Antibodies play a major role in immune responses against viruses, which inhibit infection by binding to target viral antigen. Antibodies are induced by viral entry to the body and vaccination that artificially induces immune responses; therefore, antibody tests are used in research for infection history and evaluation of vaccine efficacy. Currently, antibody tests against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by immunochromatography, enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescence immunoassay (ECLIA), or other kits used for automated analyzer are available. However, the test results should be carefully interpreted because requirements for the antibody test to obtain reliable results have not been established. Also, antibodies in human samples are heterogeneous, and their expression level changes over time. This review briefly explains the basic knowledge about antibodies against SARS-CoV-2 and outlines the classification and characteristics of the antibody tests with points to consider in their use. A summary of the collaborative study that evaluated the analytical performance of antibody test kits conducted by the National Institute of Health Sciences is also introduced. Then, the issues in ensuring the reliability of antibody tests results are discussed by considering the usefulness and availability of the World Health Organization international standard for anti-SARS-CoV-2 immunoglobulin.