BioPharm

26 BioPharm International eBook April 2019 www.biopharminternational.com Outsourcing Resources Bioanalytical Methods Flow cytometry platforms can measure multiple parameters within a single analysis, and it is a power- ful tool for cell-therapy programs. The technology allows for real-time monitoring of the cell therapy in vivo and can support the qPCR data for PK. Flow panels can be designed to evaluate the expression and per- sistence of the construct over time but can also provide insight into the immune activation status by the addition of antibodies to known markers on lymphocytes. Stability of samples need to be taken into con- sideration when performing flow for cell-based therapies. To maintain sample integrity, in most instances, samples need to be analyzed within 24 – 48 hours, which can lead to logistical challenges. The European Bioanalysis Forum released a white paper in 2017 (8) with the intent to provide practice guidance on the use of flow cytometry for regulated stud- ies that support drug development programs. The paper addresses the use of flow cytometry from a bio- analytical perspective and addresses additional parameters not discussed in previous publications. In genome-editing applications, newer, rapid-sequencing platforms such as next-generation sequencing (NGS) offer techniques to monitor both the efficacy of editing as well as potential off-target gene edits. These technologies are challenging to vali- date and therefore are typically per- formed in a non-good laboratory practice fashion. There currently are no regulatory guidance docu- ments that govern this body of work; however, as NGS is used in clinical laboratories for diagnostic purposes, published guidelines (9) governing that work may be leveraged where and when appropriate. Tried and true platforms such as ELISA maintain a presence in the gene- and cell-therapy bio - analytical space due to relatively low cost, ease of use, and solid performance. ELISA assays can be applied to the detection of anti- capsid or anti-transgene antibod- ies, and to detection of antibody responses to other components of the gene or cell therapy that may possess immunogenic potential. A requirement for enhanced sen- sitivity may benefit from newer technologies such as electrochemi- luminescence or resonance-based platforms. These newer platforms also offer the advantage of being species-agnostic and therefore can be used throughout the nonclini- cal and clinical continuum. FDA, along with other global regulatory bodies, have published specific guidance documents for the devel- opment, validation, and assess- ment of anti-drug antibodies (5). Effort should be made to adhere to these guidance documents in the assessment of immunogenicity to gene and cell-therapy elements. In some situations, the nature of the therapy, molecule, disease, or patient may dictate the need to derive a fit-for-purpose approach to immunogenicity assessment. Cell- based assays provide an essential platform for the detection of neutral- izing antibodies. Cells that are per- missive to transduction by the viral vector are exploited for this purpose in gene therapy programs. To-date, the design, execution, and reporting of data from these assays has not been standardized, making compari- son across studies challenging. Commercial ELISA kits can be used for the quantification of the newly-expressed protein resulting from gene therapy. In this setting, the measurement of expressed pro- tein is considered a biomarker, and the assay should be validated to the requirements specified in FDA's May 2018 bioanalytical guidance (2). More recent technologies such as Simoa (Quanterix) and SMC Errena (Sing ulex) instruments promise ultra-high sensitivity, which is an advantage when trying to quantify the typically very low levels of pro- tein. Recently, the market has seen an increase in highly sensitive com- mercial ELISA kits and chemilumi- nescent and fluorescent substrates that also enhance sensitivity. Other platforms can be applied to the functional assessment of expressed proteins; examples include clot- ting time for expressed coagula- tion factors or substrate cleavage by expressed enzymes. FDA released guidance on biomarker evidentiary framework (10) that describes a rec- ommended approach for analytical biomarker assays. With the newer generation and more complex gene and cell-therapy products in development, it is impor- tant to seek scientific expertise to plan and design what is needed to develop and validate the bioanalyti- cal methods required to achieve an overall successful program. REFERENCES 1. R.M. Blaese et. al., Science. 270 (5235), 475–480 (1995). 2. FDA, Guidance for Industry: Bioanalytical Method Validation (Rockville, MD, May 2018). 3. FDA, Guidance for Industry: Gene Therapy Clinical Trials—Observing Subjects for Delayed Adverse Events (Rockville, MD, November 2006). 4. FDA, Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products (Rockville, MD, November 2013). 5. FDA, Draft Guidance for Industry: Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products (Rockville, MD, April 2016). 6. FDA, Guidance for Industry: Design and Analysis of Shedding Studies for Virus of Bacteria-Based Gene Therapy and Oncolytic Products (Rockville, MD, August 2015). 7. S.A. Bustin et. al., Clin. Chem. 55(4), 611–622 (2009). 8. B.V. Der Strate, et. al., Bioanalysis. 9(16),1253-64 (2017). 9. R. Somak, et. al., J. Mol. Diag. 20(1), 4-27 (2018). 10. FDA, Guidance for Industry and FDA Staff: Biomarker Qualification: Evidentiary Framework (Rockville, MD, December 2018). BP

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