Julian Braybrook

Effective standardization strategies are critical to the successful development and adoption of complex technological solutions, in many cases due to meeting requirements of interdisciplinary expertise, interconnected systems, or the production of comparable products at scale. Cell and gene therapies, as a new generation of living drugs are a prime example of such a technology, yet with the pace of advance, the challenge to develop appropriate solutions is an increasingly complex and resource-intensive activity, requiring careful prioritization and management. This chapter introduces and develops various drivers for standardization in this sector, the stakeholders involved in this work, and governance structures required for standards development and deployment, helping to overcome challenges associated with product and process characterization, consistent manufacture and regulatory approval.

Viral vectors are agents enabling gene transfer and genome editing and have widespread utility across the healthcare and biotechnology sectors. In January 2023, the International Bureau for Weights and Measures’ Consultative Committee for Amount of Substance (CCQM) held a workshop on Metrology for Viral systems as molecular tools. The workshop brought together international leaders from across regulatory, industry, government science, and metrology sectors to better understand key challenges for the community: Exploring current limitations in the measurement of virus-derived, virus-based, and virus-like systems in terms of quantification and characterisation; surveying the state-of-the-art in analytical methods and reference material provision for these entities; and initiating a dialog for the strategic development and implementation of suitable standardisation approaches for this sector. This article presents the workshop background and rationale, presentation summaries, conclusions, and recommendations.

Flow cytometry is widely used within the manufacturing of cell and gene therapies to measure and characterise cells. Conventional manual data analysis relies heavily on operator judgement, presenting a major source of variation that can adversely impact the quality and predictive potential of therapies given to patients. Computational tools have the capacity to minimise operator variation and bias in flow cytometry data analysis; however, in many cases, confidence in these technologies has yet to be fully established mirrored by aspects of regulatory concern. Here, we employed synthetic flow cytometry datasets containing controlled population characteristics of separation, and normal/skew distributions to investigate the accuracy and reproducibility of six cell population identification tools, each of which implement different unsupervised clustering algorithms: Flock2, flowMeans, FlowSOM, PhenoGraph, SPADE3 and SWIFT (density-based, k-means, self-organising map, k-nearest neighbour, deterministic k-means, and model-based clustering, respectively). We found that outputs from software analysing the same reference synthetic dataset vary considerably and accuracy deteriorates as the cluster separation index falls below zero. Consequently, as clusters begin to merge, the flowMeans and Flock2 software platforms struggle to identify target clusters more than other platforms. Moreover, the presence of skewed cell populations resulted in poor performance from SWIFT, though FlowSOM, PhenoGraph and SPADE3 were relatively unaffected in comparison. These findings illustrate how novel flow cytometry synthetic datasets can be utilised to validate a range of automated cell identification methods, leading to enhanced confidence in the data quality of automated cell characterisations and enumerations.

Application of synthetic datasets in training and validation of analysis tools have led to improvements in many decision-making tasks in a range of domains from computer vision to digital pathology. Synthetic datasets overcome the constraints of real-world datasets, namely difficulties in collection and labelling, expense, time and privacy concerns. In flow cytometry, real cell-based datasets are limited by properties such as size, number of parameters, distance between cell populations and distributions, and are often focused on a narrow range of disease or cell types. Researchers in some cases have designed these desired properties into synthetic datasets, however operators have implemented them in inconsistent approaches and there is a scarcity of publicly available, high-quality synthetic datasets. In this research, we propose a method to systematically design and generate flow cytometry synthetic datasets with highly controlled characteristics. We demonstrate the generation of two-cluster synthetic datasets with specific degrees of separation between cell populations, and of non-normal distributions with increasing levels of skewness and orientations of skew pairs. We apply our synthetic datasets to test the performance of a popular automated cell populations identification software, SPADE3, and define the region where the software performance decreases as the clusters get closer together. Application of the synthetic skewed dataset suggests the software is capable of processing non-normal data. We calculate the classification accuracy of SPADE3 with robustness not achievable with real-world datasets. Our approach aims to advance research towards generation of high-quality synthetic flow cytometry datasets, and to increase their awareness among the community. The synthetic datasets can be utilised in benchmarking studies that critically evaluate cell population identification tools and help illustrate potential digital platform inconsistencies. These datasets have the potential to improve cell characterisation workflows that integrate automated analysis in clinical diagnostics and cell therapy manufacturing.

Measured variability of product within Cell and Gene Therapy (CGT) manufacturing arises from numerous sources across pre-analytical to post-analytical phases of testing. Operators are a function of the manufacturing process and are an important source of variability as a result of personal differences impacted by numerous factors. This research uses measurement uncertainty in comparison to Coefficient of Variation to quantify variation of participants when they complete Flow Cytometry data analysis through a 5-step gating sequence. Two study stages captured participants applying gates using their own judgement, and then following a diagrammatical protocol, respectively. Measurement uncertainty was quantified for each participant (and analysis phase) by following Guide to the Expression of Uncertainty in Measurement protocols, combining their standard deviations in quadrature from each gating step in the respective protocols. When participants followed a diagrammatical protocol, variation between participants reduced by 57%, increasing confidence in a more uniform reported cell count percentage. Measurement uncertainty provided greater resolution to the analysis processes, identifying that most variability contributed in the Flow Cytometry gating process is from the very first gate, where isolating target cells from dead or dying cells is required. This work has demonstrated the potential for greater usage of measurement uncertainty within CGT manufacturing scenarios, due to the resolution it provides for root cause analysis and continuous improvement.

Background: Flow Cytometry is a complex measurement characterisation technique, utilised within the manufacture, measurement and release of Cell and Gene Therapy products for rapid, high content and multiplexed discriminatory cell analysis. A number of factors influence the variability in the measurement reported including, but not limited to, biological variation, reagent variation, laser and optical configurations and data analysis methods. This research focuses on understanding the contribution of manual operator variability within the data analysis phase. Methods: 38 participants completed a questionnaire providing information about experience and motivational factors, before completing a simple gating study. Results were analysed using Gauge Repeatability & Reproducibility techniques to quantify participant uncertainty. The various stages of the gating sequence were combined through summation in quadrature and expanded to give each participant a representative uncertainty value. Results: 85 % of participants surveyed preferred manual gating to automated data analysis, with primary reasons being legacy (″it′s always been done that way″) and accuracy, not in the metrological sense, but in clear definition of the correct target population. The median expanded uncertainty was calculated as 3.6 % for the population studied, with no significant difference between more or less experienced users. Discussion: Operator subjectivity can be quantified to include within measurement uncertainty budgets, required for various standards and qualifications. An emphasis on biomanufacturing measurement terminology is needed to help understand future and potential solutions, possibly looking at translational clinical models to engage and enhance better training and protocols within industrial and research settings.

LGC is the UK's designated National Measurement Institute for chemical and bioanalytical measurement, and through this role improves the quality and international acceptance of measurements performed within the UK. This research spotlight, highlighting measurement 'across the scale', from elemental analysis and small molecules, through to proteins, DNA and RNA and on to whole cells and tissues, demonstrates how LGC is supporting the clinical sector by ensuring sound measurement practice that underpins clinical efficacy, quality assurance and patient safety.