Reporter gene assays that measure T-cell activation are generally faster, more consistent, and more robust than traditional cell killing assays while also reflecting a molecule’s mechanism of action. However, despite their advantages, procedures for reporter gene assays can be labor-intensive, resistant to automation, and challenging to fit neatly into an 8-hour workday. This talk describes the development and optimization of a more efficient cell-based reporter-gene potency assay to replace an analogous method used throughout Phase I clinical development of a T-cell recruiting bispecific antibody. Efficiency gains were made by evaluating alternative reporter gene cell lines, strategically optimizing individual procedure steps, and adding options for automation where possible. The new assay was shown to be comparable to the Phase I method and will be validated as the molecule’s commercial potency assay.
Protein-based therapeutics have undergone an evolution since the first products were marketed over 30 years ago. These early modalities were directed towards interaction with a single target while currently, many protein-based therapeutics are being designed to encompass more than one functional domain and are engineered to interact with more than one target. The Bispecific T-cell Engager (BiTE®) therapeutic class are comprised of two single chain variable fragments (scFv) from two distinct monoclonal antibodies which are connected by a short linker. One scFv is directed against cluster of differentiation 3 (CD3) on T cells, while the other is directed against a cell-surface tumor antigen. In vivo, the engagement of the BiTE® molecule with the T cell and the tumor cell induces the formation of a cytolytic synapse resulting in specific lysis of the tumor cells. In vitro, T cell dependent cellular cytotoxicity (TDCC) assays are used to demonstrate this mechanism of action. Traditional dual-cell based cytotoxicity assays have included labelling target cells with either dyes or radioactivity rendering the assay format to be non-homogeneous. We have developed and implemented homogeneous dual cell-based TDCC assays by employing the use of target cells that constitutively express luciferase as a surrogate marker of viable cell number. By utilizing this format for target cells, the change in viable target cell number can be readily detected by addition of a reagent that contains detergent to lyse the cells, and luciferin, a substrate for luciferase. This talk will highlight some of the important elements to consider during assay development.
Bispecific product submissions to the United States Food and Drug Administration (FDA) have increased over recent years. This may likely be attributed to advanced biotechnologies, enhanced manufacturing knowledge of therapeutic antibody products, and strong scientific rationale for the development of biologics with the ability to engage more than one target. Concurrent to the development of these complex biologic products with multiple modalities comes the need to develop bioassays that are not only accurate and reproducible, but also adequately reflective of the proposed mechanism(s) of action. Well-developed bioassays are critical aspects to the characterization and control of biological products, as well as to the interpretation of clinical study results. Bispecific bioassay development presents a unique set of challenges for assay design, such as the ability to fulfill the desired performance of the assay to capture the dual activities and potential synergist effects of the molecule preferably using a single assay format, and to detect multifaceted structural changes. This presentation will discuss challenges observed and general considerations for bispecific bioassay development from a regulatory perspective.
For over 20 years, Stegmann Systems has been developing their software solution PLA which supports the development, execution and analysis of biological assays and dose-response relationships. PLA is used by over 700 companies worldwide. Among them are the top 100 pharmaceutical and biotech companies together with laboratories, universities, research institutions and regulatory authorities. The current version PLA 3.0 is fully compliant with GAMP, GMP, and 21 CFR Part 11 and follows the guidance from the USP and Ph. Eur. PLA is the most used software for biostatistical database solutions in highly regulated pharmaceutical and biotech industries. Visit our Virtual Booth: https://www.bioassay.de/v-booth-casss-april-2020/
The number and diversity of biotherapeutic modalities continues to increase. These represent complex modalities such as peptide and peptide conjugates, antisense oligonucleotides, DNA- and RNA-based therapies, drug conjugates, as well as viral vector-based therapeutics. Biological assays are critical tools for drug discovery, development and analytical control strategies for biotherapeutics. The ability of these biological assays to reflect the relevant biological activities is essential; however, developing such assays is increasingly challenging given that these newer biotherapeutics possess a diversity of mechanism(s) of action. The session will focus on biological assays used for release and/or characterization of these emerging modalities, and the challenges to develop such assays. Talks will assess bioassay strategies, approaches and lessons learned.
Oncolytic virotherapy using native or genetically engineered oncolytic virus (OV) is a new promising strategy against tumor. It offers a multimodal approach to specifically target and effectively destroy cancer cells. The potential mechanism of action (MOA) involve direct cell killing by viral infection/replication, stimulating immune system by antigen release, transgene delivery to drive immunomodulation, etc. Due to the complexity of virus structure, manufacture process, it is critical to develop appropriate analytical and biological assays to enable process development, product characterization and quality control. Here we present an analytical and bioassay development strategy for an OV product using recombinant Newcastle disease virus (NDV). The whole analytical package contains assays for determining the identity, titer/potency, transgene expression level and functionality, as well as impurities, morphology, appearance, gene copy number, etc. These assays are used for lot release, stability study and molecule characterization. The strategy fulfilled the requirement of a thorough assessment of the product to ensure desired efficacy, safety and quality attributes.
Good Regulatory Practices are internationally recognised processes, systems, tools and methods for improving the quality of regulations. As specified by WHO, GRP aims at making sure that regulations are fit for purpose and will deliver what they are set out to achieve in terms of policy objectives. In this presentation we will review the pyramid of Moroccan legislative texts and Moroccan pharmaceutical regulations since the creation of the Moroccan pharmaceutical authority represented by the Directorate of Medicines and Pharmacy. Moroccan pharmaceutical authority adopt policies that promote regulatory convergence and harmonization (directives of WHO, ICH, EMA...), therefore, it is member of OMCLs (European Official Medicines Control Laboratory), USP associate member and European pharmacopoeia observer member. Despite the fact that the Moroccan authority has made progress in the documentary assessment of biological and biosimilar drugs, it considers that great efforts are necessary to deploy within the framework of the analytical control of these drugs and to frame the so-called drugs of the innovative therapy, namely cell therapies and gene therapies. To increase its level of competence and mastery of the drug market, the DMP has set up a new biotechnology laboratory for the analytical control of biosimilar, biotechnologies drugs and vaccines in the context of a registration and in post-marketing surveillance. The bioassay laboratory, is part of the units of the new biotechnology laboratory.