Development, validation, and application of a method for assessing the viability of Escherichia coli for flow cytometry

Introduction. About 30 % of genetically engineered therapeutic proteins are produced in Escherichia coli. In the production of these proteins, cell viability is one of the key factors determining process efficiency and product quality. Controlling viability is essential when creating cell banks and optimizing E. coli cultivation conditions. Changes in cultivation parameters can affect viability and lead to shifts in the proportions of viable cells, dead cells, and cells undergoing apoptosis-like death (ALD). Flow cytometry is one method used to assess viability and distinguish between different cell populations.

Aim. To develop, validate and test a methodology for assessing the viability of E. coli using flow cytometry.

Materials and methods. To assess cell viability, we stained E. coli with propidium iodide (PI) and annexin V-FITC (An-V-FITC). We identified populations of live, dead, and ALD cells using dual staining with PI and An-V-FITC. The method was validated in accordance with the State Pharmacopoeia, the Decision of the EEC Council No. 85 dated 03.11.2016, and ICH guidelines. We evaluated the method’s applicability in developing conditions for cell bank creation and cultivation.

Results and discussion. We developed a method for assessing the viability of E. coli cells using flow cytometry and dual staining with PI and An-V-FITC, which enables the identification of live, dead, and ALD cell populations. Validation results demonstrated that the method meets the criteria: specificity (6 %), linearity (R² > 0.9), accuracy (97–102 %), the limit of quantification (confirmed, 117 %), repeatability (1–10 %), intra-laboratory precision (2–17 %), analytical range (6.4–100 %). In selecting optimal conditions for cell bank creation, viability assessment enabled us to determine the optimal ratio of culture fluid to cryoprotectant (3 : 1) at an optical density of OD₆₀₀ = 15, resulting in over 97 % viable cells after cryopreservation. During strain cultivation process development, the method facilitated the selection of optimal conditions. In the bioreactor, the proportion of dead cells increased by only 2.5 %, and the proportion of ALD cells increased by 7 % after 8 hours of induction.

Conclusion. We developed and validated a methodology for assessing E. coli viability that can be used during the development and production of therapeutic products.

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