Harmonization in Cell Preparation: Preserving Data Comparability Across Sites, CROs, and Automation Platforms

This article focuses on harmonization at the level of cell preparation, where execution variability most often enters automated workflows.

What “harmonization” means scientifically

In cell preparation workflows, harmonization refers to the intentional alignment of execution parameters so that different operators or teams perform functionally equivalent workflows, enabling meaningful comparison of downstream data. These parameters include step order, timing, volumes, exchange behavior, and the rules that govern execution. 

Harmonization is often conflated with related concepts, but the distinctions matter: 

• Reproducibility describes an observed outcome. 

• Standardization typically refers to using the same tools or protocols. 

• Harmonization addresses the process—whether workflows are designed to be equivalent in execution. 

Harmonization does not claim to improve biology. It is a design condition that constrains variability introduced by execution, making downstream comparisons more interpretable and transferable. This distinction aligns with how regulatory, clinical, and consortium frameworks treat reproducibility as an outcome, while harmonization is treated as a design requirement [4].

Why cell preparation resists harmonization

Cell preparation is difficult to harmonize because many of its most influential variables are implicit. Manual workflows encode critical parameters—aspiration behavior, mixing intensity, dwell times, judgments about completeness—through technique rather than specification. Standard operating procedures (SOPs) describe what to do, but rarely capture how actions are physically executed. As a result: 

• Two operators following the same protocol may apply very different forces or timings 

• The same workflow may be interpreted differently across teams 

• Key execution variables remain invisible and non-transferable 

Studies evaluating aspiration speed, wash force, and resuspension intensity have demonstrated measurable impacts on cell recovery, stress markers, and population frequencies [5, 6, 7]. This is not a failure of rigor. Many preparatory techniques evolved as hands-on practices, optimized locally and passed on informally. That approach works—until workflows need to move. 

Importantly, this execution-dependent variability has been documented across cell-based assays. Studies in cytometry and assay development literature show that aspiration, washing, and resuspension steps can introduce measurable cell stress and variability even when downstream detection and analysis are well controlled.

Harmonization and reproducibility serve different roles

Reproducibility describes whether a result can be obtained again. Harmonization addresses whether a workflow is designed to produce comparable results when executed in different contexts. 

A workflow may be reproducible within a single lab without being harmonized across sites. Skilled operators and stable local conditions can yield consistent outcomes even when execution details remain implicit. When that same workflow is transferred—to another team, instrument, or site—results often diverge, not because the biology has changed, but because execution has. This is why reproducibility often fails during assay transfer—not because assays are invalid, but because execution assumptions are no longer shared [8]. 

Harmonization reduces this fragility by making preparation parameters explicit and system-defined. In this way, reproducibility becomes an outcome that can persist beyond a single lab rather than a local property dependent on technique.

What harmonized cell preparation looks like in practice

In biopharma, harmonization is not about enforcing identical protocols or tools. It is about making critical execution parameters explicit, controlled, and transferable, so assays behave comparably when run by different teams, at different sites, or on different automation platforms. 

• Defined exchange behavior – How fluid is exchanged matters as much as how much. 

Instructions such as “remove supernatant” or “resuspend cells” conceal substantial variability. Aspiration rate, proximity to the cell layer, and fluid introduction influence cell loss, stress, and assay readouts—particularly in sensitive cell-based and flow cytometry workflows [9]. In harmonized preparation, exchange behavior is defined, not implied. 

• Explicit timing and sequencing – Timing is a parameter, not an operational convenience. 

In busy biopharma labs, timing between steps varies with workload and instrument availability. Small differences in dwell time or delays between washes can accumulate into meaningful variability. Harmonized workflows preserve step order and intervals so assays remain comparable across time and facilities. 

• Fewer discretionary decision points – Reduce reliance on individual judgment during execution. 

Many preparation steps depend on operator judgment—when a wash is “complete,” how vigorously to resuspend, or when to proceed. Harmonized workflows encode execution rules directly into the process, reducing dependence on local technique and experience [10]. 

• Parameter preservation across instruments – Equivalence matters more than uniformity.

Large organizations rarely standardize on a single automation platform. Large biopharma organizations routinely operate mixed automation fleets, making execution portability more practical than hardware uniformity [11, 12]. One site may use a Biomek system while another uses Opentrons or a different liquid handler. Harmonization does not require identical hardware; it requires that defining execution parameters (e.g. CQAs, Industry 5.0 reppraisal) are preserved across instruments, so the same preparation logic produces comparable results regardless of platform.

Why automation enables harmonization across sites and platforms

Automation enables harmonization by externalizing execution parameters from individual technique into the workflow itself. Exchange behavior, timing, sequencing, and progression rules become executable parameters rather than inferred actions. 

In biopharma settings, this is critical during assay handoff, tech transfer, or CRO execution, where execution details must be specified, transferred, and audited. Manual workflows struggle to support this level of consistency at scale. 

In practice, this can take different forms. For example, software-defined approaches such as Curiox’s C-FREE Pluto Code allow centrifuge-free washing to be deployed on existing liquid handlers, rather than requiring new standalone instruments. By packaging washing logic as a software library, execution parameters can be reused across sites and applied consistently on different platforms, simplifying automation layouts while supporting harmonized execution.

Why harmonization matters for assay transfer, CROs, and analytical groups

Once assays move beyond their point of origin, the consequences of non-harmonized preparation become unavoidable. 

In biopharma organizations, this often surfaces during assay transfer, tech transfer, or CRO execution. A workflow that performs reliably within one group may show subtle but persistent shifts when executed by another team, even when the same written protocol is followed. These differences are frequently discovered only after data has been generated—during comparability studies or downstream analysis—when they are costly to resolve. 

Analytical, QC, and assay development groups are particularly exposed to this risk. When preparation depends on local technique or tacit knowledge, downstream standardization in instrumentation or analysis cannot fully compensate. 

Harmonization addresses this problem upstream. By aligning execution parameters, harmonized preparation creates a stable foundation for comparing data across sites, instruments, and partners. It allows teams to distinguish variability introduced by biology from variability introduced by execution—enabling reproducibility to persist beyond a single lab and allowing data generated across teams and platforms to be interpreted together with confidence. 

A practical next step 

For organizations exploring how harmonized, centrifuge-free preparation can be implemented without increasing system complexity, software-defined approaches offer a practical path forward. 

Curiox’s C-FREE Pluto Code, enables centrifuge-free washing on existing liquid handlers through a software library installation. By defining washing behavior at the software level, teams can share execution parameters across sites without introducing additional hardware. 

Learn more about C-FREE Pluto Code →