The establishment and management of a comprehensive cell bank system is central to biologics and cell and gene therapy manufacturing. Yet, navigating the requirements, best practices, and regulatory expectations from research through to end-of-production can be complex. In this article, we provide an in-depth look at the full cell bank lifecycle, clarifying the purpose and expectations of each bank type and highlighting critical success factors for each stage.

1. The Foundation: Research Cell Banks (RCB)

Although not formally regulated under GMP, the Research Cell Bank (RCB) plays a pivotal role in the development of cell lines used in manufacturing. It is typically established from parental or genetically engineered cells and is often the first banked version of a production cell line.

Key Practices for RCBs:

• Follow GLP-like principles: clear documentation, traceability, controlled storage.
• Confirm identity and absence of microbial contamination (bacteria, yeast, fungi, mycoplasma).
• Perform basic characterisation such as viability, morphology, and genetic markers.
• Test for adventitious agents if the cells have been in contact with animal- or human-derived components.

Importantly, even at the RCB stage, the use of serum or trypsin of animal origin introduces the need for traceability and potential risk mitigation later. If these components are used, it is advisable to retest downstream banks or transition to animal-origin-free systems early.

2. Master Cell Bank (MCB): The GMP Starting Point

The Master Cell Bank is the cornerstone of the cell banking system. It must be produced under GMP conditions and consists of a large, homogenous pool of cells derived from a single clone or cell line.

MCB Characteristics:

• Manufactured under GMP.
• Stored under defined and validated conditions.
• Subject to a full characterisation and safety testing programme.

Testing Requirements Include:

• Identity (species and cell line confirmation).
• Purity (free from bacteria, yeast, fungi, and mycoplasma).
• Safety (absence of adventitious viruses via in vitro, in vivo, and PCR-based methods).
• Genetic stability (phenotypic and genotypic characterisation).

Authorities including the FDA and EMA require comprehensive documentation of the MCB, including source history, passage number, and all raw materials used in its development. Special attention is paid to the presence of any animal-derived components and their clearance.

3. Working Cell Bank (WCB): The Workhorse of Manufacturing

Derived from the MCB, the Working Cell Bank is the cell source used to initiate production batches. While often mistaken for simply an extension of the MCB, it plays a distinct and crucial role.

Why a WCB is Needed:

• Minimises risk to the MCB by reducing direct handling.
• Supports campaign-based manufacturing.
• Enables consistency in manufacturing runs.

Testing Focus for WCBs:

• Identity, viability, and microbial purity.
• Limited viral testing if already completed for the MCB.

In most cases, full characterisation does not need to be repeated if the WCB was derived from the MCB under controlled conditions and using qualified procedures. However, in some jurisdictions or for certain products (e.g., vaccines or advanced therapies), additional testing may be recommended or required.

4. End-of-Production Cells / Cell Banks (EoPC/EoPCB)

The End-of-Production Cell Bank, or End-of-Production Cells (EoPC), represents a critical regulatory checkpoint. These cells are sampled at the conclusion of the upstream production process and are used to demonstrate that the manufacturing process has not altered the fundamental characteristics of the production cell line.

Regulatory Expectation:

• Required at the time of BLA/MAA submission.
• Demonstrates genetic stability and safety of the production cell line at its lifecycle limit.

Typical Tests Include:

• Identity and purity.
• Phenotypic and genotypic comparison with the MCB.
• Viral safety testing.

Clarification: EoPC typically refers to harvested cells taken directly from the final production run, while EoPCB refers to a formal bank of these cells, stored and maintained under GMP-like conditions. Both are used to assess genetic and phenotypic consistency, but the format chosen may depend on product and regulatory strategy.

If end-of-production samples cannot be collected (e.g., due to lysis in virus production), mock runs using uninfected cells or extended culture runs are acceptable alternatives. Regulators accept this approach when justified.

Strategic Tip: Establish the EoPC well before Phase 3 manufacturing to mitigate the risk of discovering unacceptable genetic drift or contamination late in development.

5. The Concept of the Limit of In Vitro Cell Age (LIVCA)

The LIVCA represents the maximum number of population doublings cells are allowed to undergo between the MCB and EoPC. It is essential to define this limit based on actual production conditions and to validate it with appropriate stability and comparability data.

Establishing the LIVCA:

• Determine maximum upstream process length.
• Simulate worst-case expansion conditions.
• Perform phenotypic, genotypic, and product comparability assessments.

Recommended Genotypic Tools: To demonstrate genetic stability, tests may include sequencing of coding and flanking regions, gene copy number analysis (e.g., qPCR or ddPCR), and restriction fragment mapping. These support consistency of the transgene and construct across the cell age range.

In some cases, sponsors may extend cell age to increase manufacturing flexibility. If so, new EoPC studies and comparability data will be required.

6. Lifecycle Considerations and Requalification

Over a product’s lifetime, you may need to:

• Replenish a depleted WCB.
• Establish a new MCB due to changes in manufacturing platforms or sites.

In these cases:

• Perform comparability studies.
• Consider performing another full EoPC run.
• Update regulatory filings with appropriate rationale and supporting data.

Regulatory authorities expect clear documentation and justification of changes, with evidence that they do not affect product quality or safety.

Final Thoughts: Aligning Process with Lifecycle Strategy

Each type of cell bank plays a unique and interdependent role in biologics and cell therapy manufacturing. From research through to commercial release, understanding the expectations at each stage is essential to ensuring a robust, traceable, and compliant cell banking strategy.

Early planning, strong documentation, and proactive risk management can significantly reduce delays and regulatory challenges. More importantly, they help safeguard the quality, safety, and consistency of life-saving therapies.

For teams working on new biologics or advanced therapy products, investing in a solid understanding of the full cell bank lifecycle isn’t just regulatory due diligence—it’s foundational to long-term success.

Written by Educo Life Sciences Experts, Mylène Talabardon and Hervé Broly

Mylène Talabardon – With over 20 years of experience in the pharmaceutical industry, Mylène has strong experience in process development, technology transfer and process validation. She obtained her PhD in biotechnology from The Ohio State University and her environmental engineering degree from the Swiss Federal Institute of Technology (EPFL). In 2001, she joined BiogenIdec in cell culture process department, focusing on antibody production from the lab scale to manufacturing scale.

 Hervé Broly – Starting with an engineering degree in agriculture, followed by a PhD in plant physiology,  Hervé joined the Blood Transfusion Center (Lille, France) in 1982 where he implemented a unit for the development and manufacture of monoclonal antibodies against blood groups, blood proteins and viral antigens. In 1991, Hervé took the position of Head of Process Development and Manufacturing at Sorebio (Martillac, France), a contract manufacturing organization specialized in the development and manufacture of monoclonal antibodies for clinical development. He took the lead of that company in 1998 after it was bought by Serono, a Swiss biotech company (Geneva, Switzerland) in 1994.

 This article was written from an interview and materials taken from the course, Cell Bank Establishment & Testing for Biologics, Bioassays and Cell & Gene Therapies.