Proving Clonality of Cell Lines with 99.9% Probability

Proving clonality of production cell lines is an important topic in the field of cell line development. The main regulatory authorities and organizations, such as the Food and Drug Administration (FDA) require detailed evidence of monoclonality of production cell lines.

Failing to provide sufficient evidence of monoclonality will require additional manufacturing controls, which can lead to costly delays to clinical trials or drug launch. With increasing pressure to shorten timelines and improve drug safety, Cellcavista has established technologically advanced methods using both fluorescence-activated cell sorting (FACS) and Automatic whole well imaging to ensure that production cell lines are derived from a single progenitor cell.

This article is posted on our Science Snippets Blog.

Methods for Proving Monoclonality during Cell Line Development

In order to prove monoclonality of cell lines, Cellcavista, firstly use the Fluorescence-activated cell sorting (FACS), which enables a single cell to be separated from a cell suspension and cell aggregates with a high degree or purity based on particle (means cell) sizing. Single cell cloning is performed using a FACSAria™ Fusion cell sorter after cell surface staining with DyLight 650 conjugated Protein A. The FACSAria™ Fusion cell sorter enables the singularization of cells by seeding one cell into one well (384 well plates).

The schematic below shows the single cell cloning procedure at Cellcavista:

To further support and prove monoclonality, we use automated microscopic imaging of whole wells for photo-documentation directly after the sorting. In this experiment we performed whole well imaging using Cellcavista on day 0, 1, 2 and on one day between day 5 and 7. The results below show a clone confirmed as monoclonal (A) vs a clone detected as non-monoclonal (B).

An experiment was set up using a variety of cell pools generated from Cellcavista’s CHO DG44 host cell line to evaluate the probability of monoclonality. Following the experiment, Cellcavista are able to confirm monoclonality of cell lines with a probability of ≥99%.