BioPharm

www.biopharminternational.com April 2019 BioPharm International eBook 25 Outsourcing Resources Bioanalytical Methods both to the viral vector and to the product of the transgene. Most of the vectors utilized are likely to have modifications that will trigger the host to recognize it as "foreign" and elicit an immune response that may change the clearance of the vector prior to proper transduction or pro- duce neutralizing effects. In addi- tion, many of the viral vectors may react with antibodies already present in animals and humans. To prevent these pre-existing antibodies from interfering with the program, it is recommended to pre-screen the sam- ple population, and often individu- als with high titers of antibodies are excluded from the program. The assays used to assess immu- nogenicity are standard ELISAs, cell-based assays are preferred to determine neutralizing effect. The 2016 draft FDA immunogenicity guidance (5) provides the necessary assay parameters to be evaluated, including cut point determination, intra- and inter-assay precision, sensitivity, drug tolerance, specific- ity, and selectivity. At a minimum, the screening and confirmatory assays should be performed, and when needed, the anti-drug anti- bodies can be further characterized by performing titer and neutraliza- tion assays. If the delivery mechanism is a viral vector, to determine whether the vector genome was released from the patient resulting in the possibility of horizontal transmis- sion, then viral shedding (release of the vector genome from the patient) should be evaluated to determine the possibility of trans- mission to other individuals and to the environment. A quantitative approach is recommended, and therefore, qPCR is typically used. Matrices such as urine, saliva, and nasal swabs are tested to determine whether horizontal transmission of the viral vectors occurred. A guidance for this assessment was released in 2015 by FDA (6). CELL-THERAPY ASSAYS Cell-therapy products have evolved from blood, bone marrow, and stem cells to more complex engineered autologous or allogenic immune cells to treat cancer. Cell-therapy products that meet the definition of "biologic product" require complex analyti- cal strategies and unconventional bioanalytical methods to determine distribution and persistence of the product. In the case of chimeric antigen receptor T (CAR-T) cells, the construct and viral vector used for transduction are key in determin- ing the design and type of assays required. The bioanalytical methods for these types of programs typically require flow cytometry, qPCR, and immunogenicity assessments. The first consideration is the development of reagents to be used as controls within the assays. The time upfront in production and characterization of high-quality reagents will result in reduced cost and timelines for the program. Experiments to develop the meth- ods should be carefully thought through to ensure all parameters are evaluated prior to proceeding to val- idation. High sensitivity methods are required especially when evalu- ating the expansion and persistence of the CAR-T cells. When consider- ing the development of flow cytom- etry methods to detect expression of the CAR, optimal antibody and flu- orochrome combinations are deter- mined, antibodies are titrated to maximize sensitivity and specificity, and appropriate controls are incor- porated (i.e., fluorescence minus ones, experimental procedures for lysing, washing, fixation, intra- and inter-assay precision, limit of detec- tion, inter-instrument assessments, and stability). qPCR method devel- opment and validation is similar to that described previously for gene therapy programs. Immunogenicity assay development and validation will follow the recommendations in FDA's 2016 draft guidance (5); however, one consideration is the detection of antibodies to multiple sequences within the CAR construct that likely will be considered foreign to the recipient's immune system (i.e., anti-viral protein response). PLATFORMS FOR GENE- AND CELL-THERAPY PROGRAMS The choice of platform is primarily driven by and unique to the bioana- lytical needs of the gene- and cell- therapy product under development. Specificity, selectivity, and sensitivity requirements also weigh heavily into the final decision. Irrespective of the platform, the choice of reagents has a significant impact on specificity and sensitivity, and therefore time and effort are well-spent in identifying and securing a sufficient and well- characterized supply of reagents to support the lifecycle of all bioana- lytical methods. qPCR is used to quantify a viral genome in serum, tissue, or body fluids for the purpose of pharma- cokinetics (PK), biodistribution, or vector shedding evaluations (3,4,6). It is also used to measure the expres- sion levels of transgene- expressed mRNA in target tissue for gene therapy programs. PCR is a widely used platform; however, there is a lack of specific regulatory guidance and industry standards for develop- ment and validation. The Minimum Information for P ublication of Q u a n t i t a t i v e R e a l -T i m e P C R Experiments (MIQE) guidelines pub- lished in 2009 (7) were designed to standardize nomenclature, design, and interpretation of qPCR experi- ments, and prov ide a valuable reference in the absence of other publications.

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