Quality Control and Testing
Dec 06, 2022
| 7 min read

Assessment of Extractables & Leachables From Single-Use Systems in Cell & Gene Therapy Applications

Cell and gene therapy (CGT) drug products offer new treatment options and enormous hope for patients around the world. Yet along with this promise come new and unique challenges of evaluating the extractables and process equipment-related leachables (PERLs) from single-use systems (SUS).  

This article is posted on our Science Snippets Blog

Authorities require extractables and leachables testing of any materials in the manufacturing stream that come into contact with the drug product. A number of differences between CGT and traditional biopharmaceutical manufacturing give rise to new risk assessment challenges.

Challenge #1: The Cells Are the Drug Product

While cells have long been exploited as “factories” to synthesize biological drug products, cell-based and ex vivo gene therapies are different, because the cells themselves are the product. Since cells are living entities, certain bioprocess-related extractables and leachables (E&L) that accumulate in the liquid phase or on the cells themselves can negatively impact cell health and growth characteristics, as well as critical quality attributes (CQA), and safety of the product. Downstream consequences include lower bioprocess yields, reduced product efficacy, and an increased risk to patient health. To mitigate these risks, biocompatibility tests and exposure estimations must consider both the therapeutic cells and the liquid in which the cells are suspended. 

Challenge #2: Purification Limitations of CGT Bioprocesses

Compounding the risks of E&L in CGT applications is the fact that there is essentially no downstream purification in cell manufacturing, other than a few wash steps. Downstream processes for protein-based products are very efficient in removing impurities, resulting in a highly purified drug substance.  As a result, patient exposure to process equipment-related leachables (PERLs) is low. In contrast, because of the need to preserve cell integrity, there are fewer opportunities to remove PERLs and other impurities from CGT products. 

Challenge #3: Extractables and Leachables From Single-Use Technologies

CGT applications rely heavily on SUS and devices to ensure product sterility and safety. From sample collection to cell expansion to patient delivery, CGT drug products contact a variety of single-use components and materials at virtually every stage. These include bioprocess bags, filters, bioreactors, connectors, tubing and fittings. With such a diversity of components and materials, the extractables and leachables study can become complex, and unfortunately there is no one-size-fits-all standardized approach. In addition, the trend towards smaller, fully enclosed systems—for example to produce small batches of drug product for personalized treatments|autologous therapies—means that surface-to-volume ratios are relatively high and single-use contact times are extended compared to traditional biopharmaceutical drug products. This increases the chances of E&L having an impact on the process performance, the product quality or patient health.

Challenge #4: Extrapolation and Assessment Methods for Extractables and PERLs Are Weakly Developed

The differences between traditional biopharmaceutical and CGT manufacturing processes raise important questions about the extent to which existing E&L exposure data and assessment methods can be applied to CGT applications. While significant progress has been made in defining the universe of potential extractables in single-use components, until recently there has been little attention paid to the compatibility of PERLs with human cells. Available exposure data and toxicity tests are only partially applicable to cell-based therapeutics. To address this issue, Sartorius researchers investigated two areas in particular need of further development: the generation of relevant PERLs exposure data, and the methodology for studying effects of PERLs on human cells.

Modeling PERLs Exposure in CGT Applications Using a Digital Twin

Accurate estimation of PERLs exposures—to host cells, to products and ultimately to patients—is crucial in order to compare with appropriate PERLs thresholds and limits, such as half maximal effective concentration (EC50), permitted daily exposure (PDE) and threshold of toxicological concern (TTC) values. Exposure estimation for dynamic CGT processes is a major challenge, because direct measurement of trace contaminants adsorbed on cells or dissolved in the liquid phase is extremely difficult, if not impossible. Accurately factoring in cell growth and perfusion flow rates adds to the complexity of the study.  

A cost-effective alternative to physical testing is to simulate the interaction of test compounds with cells using a digital twin. A digital twin is a virtual model of a production process based on historical and current data. To model PERLs exposure, we developed a mechanistic digital twin representing a CGT perfusion bioreactor. The model inputs included data describing specific SUS extractables and scaling, system geometry, the cell growth function, adsorption coefficients (Kd) for the dedicated cell line, and process information, such as the run time, perfusion flow rate and temperature. With this model, we were able to closely replicate the CGT production environment and predict how concentration of potential PERLs compounds would vary over time in both the liquid phase and adsorbed to the cells.  

Simulating various cultivation scenarios with the model leads consistently to similar results. In particular, the media flow rate through the system is much higher than the rate of release of PERLs from SUS components into the liquid phase. This results in a strong net wash-out effect, and therefore no accumulation of PERLs is expected in either the liquid or the cellular phase.

Figure 1. Modeling results of a PERL exposure in a CGT perfusion bioreactor.

Investigating Effects of PERLs in CGT Using a High-Throughput Cell Painting Assay

PERLs have the potential to affect many aspects of cell health, metabolism and function, yet available systemic metrics such as PDE and tolerable daily intake (TDI) do not provide insight into what is happening at the level of the individual cell.  Moreover, these values and other toxicity data are often derived from classical toxicity tests that use non-human organisms or cell lines, which may not accurately reflect human responses to test compounds. For CGT products, demonstration of the absence of any undesired effect on therapeutic cells is a requirement. Current tests that assess only one or a few end points are not sufficient for CGT applications. They can easily miss subtle changes in cell phenotype and are biased towards specific endpoints.

To address these shortcomings, we partnered with researchers at Max Planck Institute Dortmund to assess PERLs impact on living cells using a high throughput cell painting assay (HT-CPA).

HT-CPA is an unbiased phenotypic profiling technique that multiplexes six fluorescent dyes to analyze as many as 1700 features on a cell-by-cell basis using automated high-content cell imaging and analysis. The MPI database is able to link 579 cell features out of the list of 1700 features with a known effect.

HT-CPA provided a powerful and sensitive means to detect stimulating and detrimental effects using only a small number of cells and a low amount of extract.

Of 45 commonly found E&L selected for the extractables study, 40 showed no effect (above a defined threshold) on any of the 579 cell features analyzed.

Weak effects detected by four of the compounds on protein synthesis, histone deacetylase, DNA synthesis, and cholesterol homeostasis, were consistent with previously reported activity. Only one compound showed a detrimental effect on cell growth. In addition to the individual extractables also an SU filter and a tubing extract were measured, both without any effect on cells.

Together these results demonstrate the sensitivity of HT-CPA to detect potential “trouble makers” and support the conclusion that SUS are generally biologically inert and therefore well-suited for use in CGT applications.

Figure 2. HT-CPA is a powerful tool for screening and investigation of effects of PERLs on human cells

The results of these investigations into the use of digital twin and HT-CP technologies are promising. They show that PERLs exposures can be accurately modeled using existing extractables information, and that the impact of PERLs on human cells can be efficiently evaluated with modern high-throughput technology. The findings also provide reassurance that SUS are well-suited for CGT applications, and that potentially detrimental compounds can be confidently identified and eliminated from CGT bioprocesses. In the longer term, the information gained can be used to inform the manufacture of SUS components to further improve compatibility with CGT applications.

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