Ultraviolet irradiation of trypsin, lysozyme and ß-galactosidase: how does UVC affect these enzymes when used as a secondary barrier against adventitious agents?

Trypsin is one of the essential raw materials used in the manufacturing of biopharmaceutical products. As an animal derived product, it can potentially carry a serious risk of contamination with adventitious agents that can result in production shut down and lost product. To mitigate these risks, several methods are currently being used in the industry to remove contamination including physical and chemical methods. Ultraviolet-C (UVC) light is known to inactivate adventitious agents that are resistant to physical and chemical methods and could be a secondary barrier strategy. In this study, we investigated the effect of UVC irradiation on the activity and structure of trypsin. Extreme doses of UVC light were applied to trypsin using a collimated beam apparatus. The effect of UVC light on trypsin enzymatic activity was measured using a colorimetric activity assay and the effect on structure was analyzed by spectrophotometry, gel electrophoresis, and mass spectrometry. To broaden the scope, the effect of UVC light on the activity of two additional enzymes, lysozyme and ß-galactosidase, was also examined. At high doses of UVC light, changes to protein structure and protein fragmentation resulted in decreased trypsin activity. However, minimal damage was observed at doses applicable to inactivating adventitious agents, making UVC a feasible treatment for viral inactivation of trypsin products.

Evaluating ultraviolet sensitivity of adventitious agents in biopharmaceutical manufacturing.

Incidents of contamination in biopharmaceutical production have highlighted the need to apply alternative or supplementary disinfection techniques. Ultraviolet (UV) irradiation is a well-established method for inactivating a broad range of microorganisms, and is therefore a good candidate as an orthogonal technique for disinfection. To apply UV as a safeguard against adventitious agents, the UV sensitivity of these target agents must be known so that the appropriate dose of UV may be applied to achieve the desired level of inactivation. This document compiles and reviews experimentally derived 254 nm sensitivities of organisms relevant to biopharmaceutical production. In general, different researchers have found similar sensitivity values despite a lack of uniformity in experimental design or standardized quantification techniques. Still, the lack of consistent methodologies has led to suspicious UV susceptibilities in certain instances, justifying the need to create a robust collection of sensitivity values that can be used in the design and sizing of UV systems for the inactivation of adventitious agents.

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media.

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.