On 31 July 2019 Jamie Hall (project manager at Intertek Pharmaceutical Services) joined BPI for an “Ask the Expert” webinar to explore key applications and considerations for implementing flow cytometry (FC) methods in a GMP environment. This increasingly important technique can be applied to all stages of drug development in a range of applications, including cell-line identification, immunophenotyping, and cell-based potency assays. In a regulatory environment, such flexibility can present challenges. Hall explained how scientists can approach FC method development and validation amid rigorous analytical requirements.
Flow Cytometry Technology
FC provides comprehensive data by analyzing multiple parameters in fluorescently labeled suspensions of cells or particles. The cytometer channels the suspension into a stream of single particles that passes through a flow cell. Lasers of different colors excite the fluorochromes; mirrors and filters allow their detection. Through a combination of fluorescent labels, antibodies, and other functional molecules, cytometers can measure or exclude different criteria simultaneously: e.g., cell death rate, potency, specificity, and immunogenicity. Unlike microplate photometers, FC also can determine whether changes in a sample happen to individual cells or the entire population. The level of detail makes FC invaluable for analyzing critical quality attributes (CQAs), mechanism of action (MoA), and other factors that support manufacturing and regulatory reporting.
Challenges: But FC’s versatility raises regulatory challenges. Several FC assay parameters can be modified (flow rate, detector gain, number of cells collected), and because instrument performance varies by day, results can be inconsistent or too subjective to meet regulatory standards. This is a concern especially with recent changes in ICH protocols and implementation of 21 CFR Part 11. Rigorous method development is crucial to ensuring that assay steps are tightly controlled and traceable.
Control: The most important step is to establish an assay’s endpoint, controls, and gating strategies for data analysis. Fluorescence minus one (FMO) and nonstimulated controls are useful because they help set gates and reduce subjective data interpretation. Materials are important, too, and the number of available resources complicates method development. Careful antibody choice ensures desired target binding. Although compensation techniques can prevent some fluorescence overlap, judicious fluorochrome selection enables FC equipment to distinguish between markers more effectively. Reserving the brightest fluorochromes for the rarest markers is advisable. Titrating antibodies individually facilitates stain index calculations for different dilutions, enabling researchers to optimize cell staining.
Robust method development and high-performance technology can mitigate assay problems. To ensure optimal and consistent performance, Intertek uses the Becton Dickinson (BD) FACSLyric cytometer. Its setup and tracking-bead technology enable researchers to gather system performance data and catch anomalous results. Add its audit-trail capability, and researchers can lock down processes and parameters, preventing problems during method validation.
Questions and Answers
What is the typical turnaround time for FC validation? With a fully developed method, it typically takes four to six weeks. Preparation increases the effectiveness of the validation plan, analysis, and report. We provide an expedited service that takes under three weeks for a fully developed method. The method-development phase can take much longer depending on process complexity. Starting development early is crucial.
What FC assays are you running? We currently measure expression of an internal marker in response to a client’s drug. We use median fluorescence intensity (MFI) as a readout and export the data to validated SoftMax Pro software to determine potency. We had tried to develop this assay using real-time polymerase chain reaction (RT-PCR) but found that FC was more reliable.
We also are developing an assay (based on internalization of a fluorescently labeled protein) for a drug that promotes phagocytosis.
What data processing software can you use for regulated data analysis? We use the FACSSuite software included with BD’s FACSLyric system. We validated the system and found it to be perfect for our use — particularly the review and approve functions, which help control analysis. Researchers also might be familiar with FlowJo and FCS Express systems. If you are processing data for regulatory studies, it is important to ensure that the systems comply with 21 CFR Part 11 regulations.
Would a regulatory agency expect potency data from FC and an orthogonal technique? Whether to include data from one or more techniques would depend on the biomolecule in question, its MoA, and the development phase. The FC base-potency assay alone can be enough, but all those factors must be considered.
More Online
The full presentation of this webcast can be found on the BioProcess International website at the link below.