Introduction: Endotoxins are highly pyrogenic and immunogenic molecules derived from the cell wall of Gram-negative bacteria. Their detection, prevention of entry, and removal are crucial in the production of biopharmaceutical products to ensure drug safety and efficacy. Acceptable limits are strictly defined based on the drug's route of administration, with major sources of contamination including bacterial residues, water, materials, equipment, and biofilms. Purpose: In this master's thesis, we examined the impact of model endotoxin on the growth and viability of Chinese hamster ovary cells and the effectiveness of their removal through various chromatographic steps during the biopharmaceutical production of therapeutic proteins. The goal is to confirm the negative impact of higher endotoxin concentrations on cell proliferation and to demonstrate that multi-step chromatographic purification effectively ensures endotoxin levels remain below specification limits. Methods: The experiment was divided into two parts: (1) Monitoring the effect of different endotoxin concentrations on the proliferation and viability of Chinese hamster ovary cells and protein content. (2) Evaluating the efficiency of endotoxin removal across three chromatographic steps (affinity, multimodal, ion-exchange). Endotoxin levels in the obtained samples were determined using the Limulus Amebocyte Lysate test. Protein content was measured using UV spectrophotometry. The results on the impact of added endotoxin on cell growth and viability were statistically analyzed using ANOVA in JMP software. Results: Different endotoxin concentrations did not statistically significantly affect the growth and viability of Chinese hamster ovary cells (ANOVA, p < 0.005). Affinity chromatography (Protein A) reduced endotoxin below the detection limit (< 0.005 EU/mL) and achieved a logarithmic removal factor ≥ 6.2. Multimodal and ion-exchange chromatography further contributed to overall endotoxin removal, with a cumulative logarithmic removal factor of 9.4. The yields of all three chromatographic steps were consistent with process specifications, indicating that high endotoxin concentrations did not affect the evaluated process parameters. Discussion and conclusion: The model endotoxin did not statistically significantly affect the growth and viability of Chinese hamster ovary cells. Affinity, multimodal, and ion-exchange chromatography together achieved a high cumulative logarithmic removal factor, confirming that multi-step purification is effective for endotoxin removal during the biopharmaceutical process.
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