Engineering in Life Sciences最新文献

Fungal cell disruption plays a critical role in unlocking a wide range of high-value intracellular products such as lipids, proteins, pigments, and bioactive compounds. However, lysing fungal cells is far more challenging than breaking bacterial or algal cells due to their robust and highly structured cell walls. These biological barriers demand a tailored and strategic approach depending on the fungal species, cell morphology, and downstream processing requirements. This review explores the various mechanical and non-mechanical methods used to disrupt fungal cells, beyond outlining the core principles behind each method, the engineering and process factors that influence their performance are emphasized. A comparative analysis is provided, focusing on key parameters like disruption efficiency, scalability, cost-effectiveness, and environmental impact. The review also sheds light on emerging hybrid and integrated approaches, the role of pre-treatment or co-treatment strategies, and the potential for greener and more sustainable alternatives aligned with circular bioeconomy goals. Ultimately, this review aims to serve as a guide for researchers, bioprocess engineers, and industry professionals seeking to optimize fungal bioproduct extraction in a way that is not only technically sound but also economically viable and environmentally responsible, paving the way for more efficient, scalable, and sustainable fungal-based biomanufacturing.

Today, most recombinant protein drugs are produced by mammalian cells in a stirred-type bioreactor (BR). Although cell culture scale-up strategies have been extensively investigated, scale-up and switching BRs while maintaining comparable culture performance remains a challenging step. This is because the empirical correlations used to determine operating parameters are applicable only for limited situations using similar BRs across scales. In addition, a few small scale-down models (SSDMs) are able to evaluate cellular sensitivity to the shear environment of manufacturing-scale BRs. In this study, we focused on the hydrodynamic stress associated with agitation and developed an SSDM that generates high shear stress without undesirable secondary effects such as vortex formation and severe gas hold-up. In-house BRs with various scales and configurations were used for fed-batch culture of CHO-K1 cells, and their shear environment was characterized by computational fluid dynamics (CFD). Using the dry-wet approach, we found that average shear stress was well correlated with titer decrease as an indicator of culture performance. We also confirmed that the response to shear stress differs among cell lines, and that evaluation of the shear sensitivity of cells is accordingly a risk mitigation step that is required to ensure successful scale-up.

Sequencing depth is a crucial parameter for variant calling accuracy and sensitivity. The trade-off between sequencing breadth and depth is a well-known limitation in capture-based targeted next-generation sequencing (NGS). Herein, we propose a differential depth sequencing method, SPRE-Seq, to acquire different sequencing depths for different targeted regions in an NGS panel. The SPRE-Seq performance was evaluated using a panel of reference standards and clinical samples based on our custom-designed homologous recombination deficiency (HRD) assay. By applying SPRE-Seq, the effective sequencing depths of the homologous recombination repair (HRR) and HRD regions of all seven HRD reference standards met the required thresholds with only half the sequencing data volume (reduced from 12 to 6 GB). The results for the HRR genes and HRD showed 100% consistency with the expected results. In clinical samples, the effective sequencing depth of the HRR regions was significantly higher, with a sequencing data volume of 6 GB using the SPRE-Seq approach compared with 6 GB using a regular capture approach. However, there was no significant difference between a data volume of 6 GB using SPRE-Seq and 12 GB using a regular capture method. The SPRE-Seq approach was feasible and reliable for determining the HRD status and HRR somatic variants in reference standards and clinical samples at a low sequencing volume. SPRE-Seq is a reliable, feasible, and cost-effective method that can acquire an adequate sequencing depth of an NGS panel at a low sequencing data volume.

Foam formation in stirred tank fermentation processes is a well-studied phenomenon. However, foaming in shake flask cultivations is rarely considered. Non-baffled shake flasks, in particular, are generally considered to prevent foaming problems. However, under certain process conditions, foaming in non-baffled shake flasks can occur. In this study, phenomena of foam formation in shake flasks, their impact on the maximum oxygen transfer capacity (OTR_max), and experimental reproducibility are investigated. It is shown that foaming events in shake flasks can increase the OTR_max by up to threefold. This enhanced OTR_max alters process conditions and, thereby, affects the reproducibility of experiments. Foaming in shake flasks can be induced by elements such as conventional baffles or sensor spots that are used for online measurement. Moreover, a connection between the out-of-phase phenomenon and foam formation was discovered in non-baffled shake flasks. This is especially important when cultivating microorganisms at elevated viscosities. Hence, foaming in shake flasks should be considered as significantly altering process conditions, compared to non-foaming cultures. Ensuring in-phase cultivation conditions and unhindered liquid flow in shake flasks may help to avoid foaming.

Practical application: This work provides insights into foam formation in non-baffled shake flasks and its resulting implications. Foaming can increase the maximum oxygen transfer capacity and, thus, affect process conditions. The reproducibility can be severely reduced, and a comparison between foaming and non-foaming cultivations is only possible to a limited extent. Foaming can be induced by baffles or internals, such as small sensor spots, used for online monitoring. Additionally, foaming can be caused by the out-of-phase phenomenon. This is of particular importance when cultivating microorganisms at elevated viscosities. This paper is intended to raise awareness of the topic of foam formation in the shake flask and help to correctly interpret this phenomenon.

Fixed-bed bioreactors for anchorage-dependent cells are an obvious choice for development because of their large-scale capabilities, allowing manufacturing with reduced cost and footprint. In this study, a serum-free production process for Japanese encephalitis virus (JEV) in a single-use fixed-bed bioreactor was developed and compared to conventional roller bottle production as a productivity benchmark. After optimization of serum-free cell culture conditions, an initial media screening in roller bottles showed a strong impact of growth and production media on virus yields. Selected optimized medium combinations were assessed in roller bottles and the fixed-bed bioreactor. Both systems proved to be excellent production systems for JEV, but media choice was key to achieve the highest titers. In particular, DMEM with its enriched glucose content beneficially affected viral yields, enabling potential large-scale manufacturing using the fixed-bed reactor with serum-containing or serum-free media.

Practical application: Data presented in this work show feasible ways of serum-free virus production with Vero cells, a common cell substrate in vaccine development. The fixed-bed bioreactor process described here could facilitate manufacturing activities to reduce cost and footprint while simultaneously achieving higher process control compared to conventional manufacturing systems like roller bottles. With a much better upscale potential (up to 500 m²) the fixed-bed bioreactor showed comparable or better yields to roller bottles depending on media used, even with serum-free media. This research article further emphasizes the need to optimize cell culture media or media combinations for each virus individually to achieve the highest titers. As shown, performing a simple media screening experiment to optimize yields early in process development could lead to better productivity, with a high business impact in later development stages.