Q+A on Critical Reagents Usage in Bioanalytical Labs

Critical reagents for ligand binding assays (LBA) are essential components of bioanalytical testing. These include but are not limited to antibodies used as positive controls, capture, or detection reagents; peptides as drug targets; reference standards as test analytes for pharmacokinetics; positive controls for immunogenicity studies and matrices.

Critical reagents are crucial for assay performance and data comparability across different laboratories and countries since they directly impact the results of an assay. Thus, they need to be well characterized and validated for the proper execution of preclinical and clinical studies. Critical reagent stocks must also be maintained, and their sources recorded. Below is a snapshot of some of the key questions asked on the webinar, General Practices for Critical Reagents Usage in Bioanalytical Labs.

In the following Q+A, Dr. Santosh Shah, Director of Biologics, answers key questions on critical reagent usage in bioanalytical laboratories.

What approach would you use to bridge reference standards if you want to compare head-to-head, old, and new curves and full complement of quality controls (QC): LLOQ, LQC, MQC, HQC, ULOQ?

We set up two sets of plate controls (i.e., one set = one set of standards and two sets of QCs) each prepared with two different lots separately, and check the following results:

For each set, if standards and QCs meet the acceptance criteria.

If one lot of prepared QC meets the acceptance criteria in comparison to another lot of prepared standards.

Preparing from an old and new batch of reference standards will not fit on a 96-well plate, if there is the need to include three replicates of each. Is just running one or two replicates a valid approach so all five levels of QCs can be tested?

Our most common practice is to use two replicates. If three replicates are required, we use two plates to complete the comparison of reference standards.

Do you have a standard expiry assigned to different types of critical reagents, since often the stability is not known initially? Would you be willing to share your approach for being able to assign the expiry or retest/recertification dates initially?

For critical reagents acquired from external sources, we use expiry dates from certificates of analysis (COAs) provided by the sponsor/vendor. For critical reagents prepared in-house, we usually follow our standard operating procedure (SOP) based on the type of reagent, e.g., for an antibody-based critical reagent, we assign a 1-year expiry and retest annually.

How do you apply the correction factor, as you see >20% QC bias between using two different lots of reagents? In other words, will the correction factor be applied to assay modification or study data?

The correction factor is usually used when bridging sample analysis kits, in which standards and buffers are included for standard calibrators and QCs preparation. From kit to kit, the buffer and standard material are different to a certain extent and the overall comparison in QCs will be greater than individual standard material lots. The correction factor will be applied to the study data, not the assay.

Can you speak on extending expiration dates via retesting?

We perform one run with one set of standards and two sets of QCs (HQC, MQC, and LQC) loaded to plates in duplicate. If the run passes, the expiration may be extended for a time equal to the original time interval.

What is the purpose of BIO-006 Change Control Procedure?

BIO-006 Change Control Procedure SOP provides a procedure for managing document and equipment changes at Frontage Laboratories. It outlines the method of documenting and controlling changes to issued documents and equipment, as well as tracking changes.

What should the re-testing frequency for in-house conjugated reagents, e.g., biotinylated drug or digoxin drug, be?

We usually retest once a year since the labeled reagents tend to become aggregated, leading to a higher signal over time. This change is more detrimental to ADA studies in which the raw data are evaluated for positiveness and titer of the ADA confirmed. We would rather re-label reagents than re-testing the regents.

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• Watch the complete webinar: General Practices for Critical Reagents Usage in Bioanalytical Labs.
• Watch the industry’s leading bioanalytical experts discuss the trends in LBAs and critical reagents in our panel discussion: Utilizing Ligand Binding Assays (LBA) and Critical Reagents for Drug Development.

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Frontage’s biologics teams have nearly 15 years of experience in complex drug development and large molecule applications throughout its evolution in product development. We have handled projects for peptides, proteins, monoclonal antibodies, bispecific antibodies, biosimilars, oligonucleotides, biomarkers, and antibody-drug conjugates. Contact our sales team for your biologics projects.

Note: This feature includes the webinar, panel discussion, and Q+A, and was done in association with Bioanalysis Zone.

In this feature sponsored by Frontage with Bioanalysis Zone, we interview key figures from pharma, CROs and academia about their experiences and opinions on the utilization of flow cytometry in cell and gene therapy developments.

How has flow cytometry recently contributed to the field of cellular and genetic therapeutics?

Flow cytometry is a powerful tool used in the research and development of cell and gene therapy products. With this tool, the researcher can gain valuable insight into the phenotype and function of populations of individual cells and how those cells respond to perturbations in their respective environments. In the development of cellular medicine, flow cytometry is used for the assessment of culture health, phenotypic characterization of in-process culture and final product, as well as the functional characterization, to quantify the effect of potential process changes as well as indicate the labs’ capability of making a safe and effective product on the lab bench.

Flow cytometry has been utilized for the immune monitoring of CAR-T cells. What are the next cellular targets for therapy that can be monitored with flow cytometry?

CAR-T cell characterization, such as phenotyping and functional analysis: In vitro CTL assay: CD107A Flow assay and IFN-g production.

What are some important aspects to consider when deciding on bioanalytical techniques during cell and gene therapy studies?

• Skill and Capacity Shortages: the required expertise in molecular biology or performing flow cytometry at the standards required for regulatory approval.
• Innovation and Creativity are Required
• Reagent Quality Must be Addressed. One of the key challenges facing modern bioanalytical scientists is the variability in reagent quality. For both ELISA and cell-based assay reagents, a lot of time and effort is wasted on ensuring that lot-to-lot variability does not impact the results generated over time to support a program.

How does the quantitation of cell populations aid in the development of cell and gene therapies?

According to cGMP regulations, quality is built into the design of the process and in every manufacturing step. Due to the complex nature of cell and gene therapy products, a cautiously devised list of in-process and release tests is required to provide adequate evidence of identity, safety, purity, and potency. Take CAR-T cell productions as an example, the identity of CAR-T cell products is commonly characterized by CAR surface expression. The purity of the product relies in part on specified levels of CD3+ and CAR+ T cells. Up to now, the potency of CAR-T cells is often determined by in vitro cytotoxic T lymphocyte assay or interferon-σ secretion.

What capabilities does flow cytometry bring to cell and gene therapy development that make it a favorable bioanalytical tool in the laboratory?

Just 15 years ago, an average bioanalytical lab largely relied on chromatographic methods. With the advent of mAb therapies, ligand-binding assays for immunogenicity (e.g., ELISA) became widely used. Today, cell-based assays, flow cytometry, and molecular biology-based methods, such as branch-chain DNA analysis, are important. 

Flow cytometry has itself evolved to meet changing needs. Initially designed to detect cell types based on surface characteristics, flow cytometry is now combined with the detection of specific intracellular properties (intracellular flow cytometry) to characterize signaling networks at the single-cell level. Gating strategies required to identify the cell populations are developed by the bioanalytical scientist and must be implemented in a very manual process. Flow cytometry thus involves art as much as science and requires deep knowledge and understanding of the technique and the products under evaluation. One of the key workflows is centralizing the data review, and processing to a single team for a global trial can ensure consistency in the data. The European Bioanalysis Forum document on best practices for flow cytometry in a regulated environment provides invaluable guidance on traceability and comparability of data. 

What are the bioanalytical challenges to implementing flow cytometry into cell and gene therapy developments?

Complicating the situation is the lack of specific regulatory guidance on cell-based assays and the use of flow cytometry for drug development applications. Guidance documents exist for chromatography and ligand-binding assays, but only a few white papers have been published on bioanalysis for cell and gene therapies. Regulators want to drive approvals of novel treatments, and in the absence of clear guidance, they will accept new methods provided that evidence shows that the treatments are robust and appropriate. 

Both pharma companies and CROs must be innovative and develop techniques that will enable them to provide the required data and find solutions to new challenges — such as the cross-validation of a flow cytometry method in two laboratories — as they arise. Bioanalytical scientists working with clinicians can effectively solve problems. Because no one in the industry has long-term experience working with these methods, it is vital that the bioanalytical scientists that have made progress share their insights with others for the further advancement of techniques.