Robust cell-based potency bioassays are critical to meeting global regulatory requirements for successful drug clinical studies and the commercialization phase. These assays, which include in vitro cell-based assays, must provide precise and reliable measurements of therapeutic agents, biomarkers, and pharmacokinetic parameters throughout preclinical and clinical studies.

What are phase-appropriate assays?  Phase-appropriate assays refers to specific requirements that the bioassay meets for each phase of drug progression from preclinical to commercialization.  There are 3 stages of assay development: (1) demonstration of initial fit for purpose (supports preclinical and clinical Phase 1, (2) qualification(supports clinical Phase 2 and process development), and (3) validation (supports clinical phase 3 and commercialization, although a validated assay can be used for an earlier clinical phase).

This phase-appropriate bioanalytical assay is essential to ensure that each phase of drug progression, whether Investigational New Drug (IND)-enabling studies, or pivotal clinical trials, is supported by assays tailored to their specific regulatory and scientific needs, including Quality Control (QC) and Quality Assurance (QA).  Assays must be developed in strict accordance with FDA and ICH guidelines, with an emphasis on sensitivity, specificity, accuracy, and reproducibility (precision). Adhering to these standards is vital for maintaining data integrity, ensuring compliance, supporting product and process development, and enabling the smooth progression of regulatory submissions. This report describes and incorporates the three stages of bioanalytical assays and their relationship to each phase of drug progression.

Stages of Assays Appropriate for Each Clinical Phase 

Phase Appropriate Assays: The Key to Regulatory Success

Phase-appropriate assays refers to the development, qualification and validation of a bioanalytical assay to meet the specific scientific, regulatory, and business requirements of each distinct phase of clinical drug progression (development / preclinical, clinical Phase 1, Phase 2, Phase 3, and commercial phases).

The intent of phase appropriate bioanalytical assay method development is to ensure your assay will support its intended use.  MarinBio has developed a tried-and-true framework to ensure that a specific assay is appropriately developed to meet the specific requirements of its intended use during all the steps of drug progression up to and including commercialization. This regulatory compliant framework has been used to develop multiple bioanalytical assays.  The assays are specifically tailored to an individual client’s needs, and are aligned with ICH Q14 guidelines on Analytical Procedure Development and ICH Q2R2 Validation of Analytical Procedures.

Are you at the PRE-IND Phase?

Stage 1 – FIT FOR PURPOSE: Assays for Preclinical and Clinical Phase 1 Studies

Setting up the Initial Assay: Tech Transfer or De Novo Development

2-6 experiments are required to meet Fit-for Purpose Assay Standards 

At MarinBio, we apply a consistent and efficient approach to the development or improvement of in vitro assays, whether we’re transferring an existing technology (Tech Transfer) or creating a new assay from the ground up. For technology transfers, we precisely replicate the original method, provided it is well-documented to ensure continuity and reliability.  We frequently transfer existing potency assays from our clients.  We are also experts in building assays from literature references, commercial kits, or entirely novel concepts (de novo development).

For de novo development, our first goal is to get the assay “up and running.” This often includes generating a research cell bank from which frozen cell vials will be prepared and used consistently to ensure control across the lifecycle of the assay.

Every assay development project moving toward qualification or validation begins with a strategic evaluation of key variables. Our iterative method allows us to test multiple parameters in parallel, identify optimal conditions quickly, and refine performance by revisiting earlier variables as assay methods evolve. This approach accelerates timelines, minimizes the number of experiments, reduces overall costs, and ensures a scientifically robust, well-performing assay.

Assay Development with Regulatory Readiness

Once the foundational assay is in place, it is refined and improved to be “Fit-for-Purpose” for preclinical and early clinical use. While not yet fully qualified or validated, these assays are designed to meet the expectations of FDA review for initial IND submissions. Our cell-based assay development focuses on reproducibility, accuracy, and robustness, all essential qualities for advancing to later-stage qualification and eventual GMP validation.

Are you Ready for Your Next Regulatory Step?

In U.S. drug regulation, different FDA applications are used to gain approval to study or market small molecules and biologics. The type of application depends on the drug type and phase status.

• IND (Investigational New Drug): Authorization to begin clinical trials in humans after preclinical testing.
• NDA (New Drug Application): Request to market a new small-molecule drug after demonstrating safety and efficacy in clinical trials.
• ANDA (Abbreviated New Drug Application): Application to market a generic drug by showing it is bioequivalent to an approved product.
• BLA (Biologics License Application): Submission for approval to market a biologic product, such as a vaccine or monoclonal antibody, by proving safety, purity, and potency.

Stage 2 – QUALIFIED:  Assays for Clinical Phase 2 Studies

Should you Select a Qualified or Validated Assay?

Following the initial method development, the assay should consistently measure bioactivity (e.g. potency) in an in vitro cell-based assay.  As development advances into the clinic, the focus shifts to demonstrating a repeatable and robust dose-dependent response, while ensuring acceptable intermediate precision and accuracy across multiple runs.  During this qualification stage, the assay is strategically improved to support Phase 2 clinical studies and process development. These enhancements establish a solid foundation for a robust and reliable assay to be validated for Phase 3 clinical studies and commercialization of drugs.

The Qualified Assay

3-8 experiments are required to meet Qualified Assay Standards

The goal for this stage is to qualify the assay. This involves evaluating and refining several critical performance attributes, including robustness, accuracy, precision, linearity, range, specificity, and stability (freeze/thaw), to ensure that both system suitability criteria as well as the acceptance criteria are appropriately defined. These criteria will ultimately support successful assay validation.  Assays are qualified in alignment with ICH guidelines, such as ICH Q2(R2) and FDA requirements outlined in the Analytical Procedures and Methods Validation for Drugs and Biologics Guidance Document.

At this stage, MarinBio scientists will also consult with clients about the long-term storage of Reference Standards (RS) and test samples. Typically, single-use aliquots of the RS are produced to support Phase 2 clinical studies and the eventual validation assays used to support Phase 3 clinical studies and commercialization.

Key assay parameters under investigation include identifying the ranges for cell concentration, passage number, timing, buffer systems, drug concentration, temperature sensitivity, and specificity (e.g., negative control response). This stage results in a Qualified Assay, a reliable assay with well-supported acceptance criteria needed for the later validation of the assay.

Assays Supporting Clinical and Process Development 

A minimum of three replicate experiments following the finalized assay protocol are performed to establish preliminary acceptance criteria and assay performance metrics. For a more robust qualification, two analysts typically conduct at least three independent runs, each using three assay plates. This allows for statistical analysis (e.g., relative potency and percent coefficient of variation, or %CV).

At this point, MarinBio recommends creating a 100-200 vial Master Cell depending on long-term goals and regulatory requirements. The Master Cell Bank is produced under GMP guidance with complete GMP QC/QA supervision.

Upon completion of this step, the assay may be carried out in two ways: as a qualified assay where MarinBio provides a qualification report, or MarinBio can provide full GMP documentation, including SOPs, workbook, Certificate of Testing that is formally Quality Controlled and Quality Assurance reviewed. Clients can choose either option based on their needs and the intended purpose of the assay.

Assay Acceptance Criteria

The following assay metrics are defined during assay qualification.

Stage 3 – VALIDATED: Assays for Clinical Phase 3 Studies and Commercialization

The GMP Validated Assay

6-12 experiments are required to meet GMP Validated Assay Standards

Prior to BLA/NDA submission, the assay must be fully validated and demonstrate statistical reproducibility, robustness, and minimal variability across different analysts, facilities, equipment and over time. Aligned with regulatory guidance, MarinBio recommends performing this step as early in the process as possible often before Phase 3 clinical trials begin.  These assays are performed under GMP conditions with complete documentation and oversight from Quality Control and Quality Assurance following the prevailing FDA and ICH guidance documents. Once validated, the assay must perform consistently, demonstrating it is robust, accurate, specific, and precise, supporting analysis of both activity (e.g. potency) and long-term stability of test samples.

By this stage, the assay is well-characterized. A detailed Standard Operating Procedure (SOP) is used, enabling any qualified operator to execute the method reliably. A comprehensive multi-page workbook documents all essential details, including Reference Standard and Test Sample identification, equipment used, reagents, methods (including time and temperature ranges where appropriate), as well as raw and analyzed data. Review by our QC and QA staff and signatures are required throughout the process.

A validation protocol is created outlining the planned validation experiments and acceptance criteria. This protocol is shared with our client, for review and approval, requiring signatures from both our clients’ team and MarinBio. At least two analysts are necessary, each performing at least three experiments for a minimum of six. More than six experiments are typically needed to achieve strong reproducible statistical confidence given the inherent variability of in vitro assays. These experiments also establish numeric performance criteria to guide future use of the assay for sample analysis to aid in identifying out of trend and out of expectation results. Upon completion, a Validation Report with Quality Control and Quality Assurance oversight is issued to document the successful assay validation.

Summary – Guidelines for Validation of a GMP level Assay

A summary of guidelines and minimum recommendations for validation of GMP potency cell-based assay is shown in the following Table.

Applications for a Validated Bioanalytical Assay

A validated bioanalytical assay is essential for ensuring consistent and reliable measurement of a drug’s potency. Key applications include lot release testing to confirm product quality and potency, stability studies to monitor drug performance over time, and pharmacokinetic (PK) and toxicology (tox) studies to assess drug behavior and safety in biological systems. Additionally, validated assays support regulatory submissions, comparability studies, and ongoing quality control throughout the drugs lifecycle.

Further Reading

1. FDA Analytical Procedures and Methods Validation for Drugs and Biologics; Guidance for Industry
2. FDA Bioanalytical Method Validation Guidance for Industry
3. ICH Q2(R2) (Validation of Analytical Procedures)
4. ICH Q14 (Analytical Procedure Development)