Clonally-Derived Workflows: The Need For Wrap Around Data Packages For Regulatory Submissions

Contributed Commentary by Duncan Borthwick

July 7, 2021 | A flask of cells: a biological factory. This culture could generate the next blockbuster antibody, or viral vectors to deliver gene therapies to patients, or, in the new world of stem cell therapies, be enroute to become cancer killing cells. Although the end products are diverse in purpose, they all share a need to demonstrate “clonal derivation” as part of their development process.

The ‘What’ And ‘How’ Of Clonal Derivation

Clonal derivation refers to the process by which a pool of cells is separated out to individual single cells (single-cell seeding) and then grown to generate new pools of cells from the original clones. The nature of biological variation, along with variations in the transformation process, means that the cells in the original pool will all be genetically and phenotypically different, will vary in their ability to produce the desired product at the desired scale and may respond differently to the pressures of production scale-up. As such, the clonal derivation process is there to manage heterogeneity within a cell bank and reduce variation.

Projects start out with thousands of single cells (often plated out in 96 or 384 well plates) which are pressure tested in the pursuit of lead candidates. Following single cell seeding, growth, and selection, the cells are ultimately stored in a Master Cell Bank (MCB), providing a repository of well characterised cells. This Bank allows the inherent variations of biology to be best managed, and will subsequently become the master copy of ongoing production.

Put more explicitly, minimizing the heterogeneity with the MCB helps ensure consistency in product manufacture and best manages selective pressures placed on cell cultures by changes to the upstream process, which can result in changes to the manufacturing process or the final product.

Validation and Verification

As part of regulatory submissions, including the filing of a new therapeutic antibody, viral vector, or cell therapy, commercial labs are required to demonstrate that their clonal-derivation process is sound and that the high-value master cell submitted, did indeed, start from a single cell.

Delivering a process validation report, which openly probes and embraces weaknesses in the total process and in the individual cell, requires the lab to step back and view the operation as a whole. You identify a round object in a well, the seeding method says it’s a single cell, but what confidence can you demonstrate to assign a label of ‘single cell’? You need to ask yourself: is there an extra cell hiding in a pipette tip? How often is it not a single cell? What factors affect that error? Is that small round object, that looks like a single cell dividing every 24hrs in a manner that is consistent with it actually being a cell? How would you know if two cells are dispensed into a well? How can the choice of plastics affect the quality of the process. Transferring cells between vessels, how do you avoid mis-tracking? Using plastic consumables, how do you manage contamination? The list is extensive.

Evidencing Clonality And Data Assurance

Process validation (is the process fit for purpose?) and subsequent verification (is the process still fit for purpose?) becomes the backbone of clonal assurance. There is wide diversity in how individual labs tackle this question. Historically, statistics and probability was a primary tool i.e. there should only be one cell in the well, but since the advent of bespoke imaging approaches to clonality, quality evidence is the preferred approach. Seeing a crystal clear image of a single cell in an entire well and nothing else is a comfort to both labs and Regulators.

The use of multiple points of quality data strengthens the evidence. This can include, firstly, an image of the cell immediately post dispensing in the dry well then secondly, after the media is added, a whole well image. Daily imaging to show the cell divide 1 to 2, 2 to 4, 4 to 8 and so on further adds to the case.

Partial well scanning, or imaging a cell just in a nozzle and not once it has been dispensed, detracts from confidence, as does the use of multiple separate and unconnected methodologies. In terms of risk management, the achilles heel to many operations remains uncontrollable system; operations that rely on people cutting and pasting data, spreadsheet maths and dare I say, USB sticks walking down corridors are difficult to risk manage

Getting The Part 11 Mindset

Although many of the workflows mentioned above are deemed, ‘research’ and not ‘manufacturing’, the principles and benefits of good data auditing, as described in 21 CFR Part 11, are applicable. Managing data security and auditing as to who, what, where and why all contributes to a healthy Part 11 mindset.

Having single instruments that perform multiple tasks (i.e. seeding plus imaging for assurance) and directly linking instruments that perform seeding, imaging, and selection within the same data bubble, all reduce the risk management burden.

Clonality Report For Regulatory Submission

Ultimately, the question becomes one of solid data and communication confidence: accumulating evidence in the form of high quality annotated and manually verified images plus project information within an audited environment. A quality clonality report, rich in quality evidence, that gathers up the whole story of the clonality workflow is central to Regulatory submissions.

If you consider best selling monoclonal antibody, Humira, which generated nearly $20 billion in revenue in 2018 alone, the value of getting clonality-derived workflows right, can be very high indeed. The risk of getting it wrong, well, you work out the math

Duncan Borthwick has a PhD from University of Edinburgh and University of North Carolina, Chapel Hill and since hanging up his lab coat 20+ years ago has worked in the development of novel instrumentation and products. He heads the Product Management group at Solentim working closely with the R&D, biology and commercial teams working to define the next set of products to support the cell therapy community. He can be reached at duncan.borthwick@solentim.com.