Assurance, Validation and Verification in Clonality
Duncan Borthwick PhD
This document represents the opinions of Solentim Ltd and should not be used in place of regulations or published guidance from the USFDA or any other regulatory body
Within the cell line development community there is a widely accepted regulatory expectation to demonstrate that a Master Cell Bank (MCB) has been derived from a single cell to best manage the quality of the biotherapeutic produced1,2,3. The term ‘clonally-derived population’ is a contemporary description, embracing the inevitability of genetic and phenotypic heterogeneity in the population4, particularly for CHO cells.
There is no official published methodology on how to demonstrate clonality either for quality purposes or for an IND or BLA filing, rather, it is left largely in the hands of cell line development scientists to interpret guidance and to build a process, incorporating probability, supported by well-considered data as part of their assurance package.
- Assurance: “an assessment of all the information provided, including probability calculations and supplemental data/information”5
- Probability: “a numerical calculation provided by the sponsor”5
Assurance is about understanding and managing risk. To quote Welch and Arden, “the resulting risk should be necessarily mitigated by an augmentation of the control strategy”4. In the era of limiting dilution, where a specific concentration of cells was pipetted onto a plate, understanding the risk of how many wells might have zero, one, or more than one cell, was the subject of probabilistic statistics matched with laboratory findings. This area is further confused by the observations that the number of cells in any given well affects the resulting outgrowth. Plates with low numbers of colonies are therefore less likely to be clonally derived than the statistics might suggest.
“Imaging technology oﬀers an attractive way of providing supportive data to assure clonal derivation of production cell lines in lieu of additional laboratory work” 4
It was only with the adoption of imaging-based approaches that the differences between probabilistic calculations and real-life were observed.
In 2014, Solentim launched the Cell Metric®, a high contrast bright field imaging system specifically designed to provide clear evidence around the presence of a single cell in a well. The system enables users to image wells post seeding (‘day 0’) then at daily intervals going forward as cells divide and grow into clones. Of equal importance, the instrument is there to identify the occurrences when there are no cells or multiple cells in the well. A timeline of evidence is generated automatically by the instrument, pulling image data and user annotation into a pdf. Moving from pipetting to imaging enables the discussion to move on too, from one of probability to one of validation and verification.
“One cannot demonstrate clonality, rather, one can use data to show that your cell line development methods can assure that your MCB has a high probability of being clonally derived” 6
To advance both the workflow and assurance package, the VIPSTM was launched in 2018. This ‘verified in-situ plate seeding’ system combines a low-pressure single cell seeder with immediate ‘in well’ imaging of the dispensed droplet carrying the cell. The evidence of the cell in the well combined with day 0 imaging (post media fill) produces a ‘double lock’ of evidence that a single cell was dispensed, existed in the well and grew into a colony (figure 1).
This VIPS droplet image also largely eradicates incidence of ghost wells, where a colony forms from a well with no detected single cell on day 0.
Figure 1: ‘Double lock’ of assurance workflow based on post seeding, in well image then post media fill, day 0 image.
Applying a critical multi-stage validation approach enables Solentim users to confidently prepare data packages for regulatory submission.
This, combined with verification confirmation of correct usage and service status, has enabled rapid workflow integration for new laboratories setting up for the first time and for existing or adapting workflows to de-risk the IND submission process.
With over 10 years’ experience, Solentim continues to drive assurance-based workflows for cell line development.
“The cell line generation report (or clonality report) has satisfied our IND filing requirements for the Chinese NMPA.” Andy Tsun, PhD, Co-Founder and VP of Discovery Biology, Biotheus Inc.
“The Cell Metric clonality reports are a critical component of our CLD workflows. Following cell line development custom service projects, we provide these reports directly to our customers for use in their IND filings.” Amber N. Petersen, Ph.D., Principal R&D Scientist, Millipore Sigma
1 International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Q5D: Derivation and characterization of cell substrates used for production of biotechnological/biological products. 1997.
2 US Food and Drug Administration. Points to consider in the manufacture and testing of monoclonal antibody products for human use. 1997.
3 European Medicines Agency: committee for medicinal products for human use. Guideline on development, production, characterisation, and speciﬁcation for monoclonal antibodies and related products. 2016.
6 Regulatory Considerations in Establishing Clonality for Cell lines expressing therapeutic proteins, Audrey Jia, 5th Annual Cell Line Development & Engineering Asia, May 16-19 Shanghai, China