Abstract Vero cells (referred to as “Vero” henceforth) are a commonly used host for the production of a wide variety of vaccines, but due in part to the typically anchorage‐dependent nature of the cells, Vero cells also pose multiple challenges during process development, scale‐up, and commercialization. The adaptation of Vero to proliferate in a serum‐free suspension culture while remaining non‐tumorigenic can be a challenging endeavor, and where successful, has often utilized undisclosed and/or complex custom‐designed medium formulations. Additional and expanded options for commercially available serum‐free media that can support the robust expansion of suspension‐adapted Vero cells for viral vaccine manufacturing would be a significant advantage for increasing the industrial adoption of suspension Vero, as virus production can also be highly medium‐dependent and sensitive. In this work, we have successfully transitioned suspension‐adapted Vero cells from a proprietary medium to a commercially‐available base medium (ExCell VPRO) with supplementation of additional commercially‐available components, and subsequently infected the resulting ExCell VPRO cell bank lineage with an attenuated vaccine strain Mumps virus. Like many suspension cell line media formulations, off‐the‐shelf commercially available ExCell VPRO medium contains dextran sulfate, which serves to minimize cell clumping, but is also well‐known to directly impact the infectivity of some viruses. Unexpectedly, we have observed that the presence of dextran sulfate in the infection medium increased both culture longevity and infectious virus titers for attenuated Mumps, potentially eliminating the need for a medium exchange between cell growth and infection for a more streamlined and easily scalable attenuated Mumps virus production process.
Abstract Production of human papillomavirus (HPV) virus‐like particles (VLPs) in Komagataella phaffii is often constrained by methanol‐associated toxicity and suboptimal feeding strategies during induction. In this study, a microbioreactor‐based approach was employed to systematically design and evaluate mixed‐substrate fed‐batch strategies for enhanced HPV16 L1 VLP production. Four methanol‐based feeding compositions, including methanol‐only and combinations with glycerol and sorbitol, were investigated under pulse and linear feeding modes. Among the tested conditions, a linear feeding strategy using 70% methanol and 30% glycerol (70 M + 30G) resulted in the highest biomass (DCW: 90.36 g/L) and VLP titer (42 mg/L), corresponding to a two‐fold improvement over methanol‐only feeding. Enhanced performance was attributed to reduced methanol‐induced metabolic stress and improved substrate utilization, leading to more stable dissolved oxygen (DO) profiles and sustained cellular activity. In contrast, pulse feeding resulted in significant DO fluctuations, indicating transient metabolic imbalances and lower overall productivity. The optimized feeding strategy was successfully translated to a 1 L stirred‐tank bioreactor, achieving a DCW of 120 g/L and a VLP titer of 49 mg/L, with a ~ 2.3‐fold increase compared to the control. The consistency between microscale and bench‐scale results demonstrates the predictive capability of microbioreactor systems for fed‐batch process development. Overall, this study presents a rational and scalable feeding strategy that improves recombinant VLP production while reducing methanol usage, providing a practical framework for efficient bioprocess design in K. phaffii .
Upstream continuous manufacturing of biologic therapeutics has been an attractive method to address growing demand. However, maintaining high cell densities (>100 million cells/mL) for long periods of time (>25 days) requires large amounts of media, complicating logistics and presenting a roadblock to increased adoption. To reduce volumetric media use, intensified media recipes have been developed. This study evaluates two such recipes to reduce perfusion rate by up to 30%: the first, a concentrated media formulation to increase nutritional depth, and the second, a commercial perfusion rate reducing supplement. We observe how the cell process, productivity, and product quality are impacted. Direct media intensification stalls cell growth by increasing osmolality, ultimately decreasing cell viability. Meanwhile, media with the perfusion rate reducing supplement maintains cell viability and productivity and has no significant impact on product quality compared to the control. We demonstrated the generalizability of the perfusion rate reducing supplement by testing with a different media and cell clone and observed similar results. Cell cycle analysis demonstrated that both intensified media strategies successfully stall cells in the G0/G1 phase, but the perfusion rate reducing supplement more effectively arrests cell proliferation, suggesting a larger shift from growth-driven cells in glycolysis to production-driven cells in oxidative metabolism, and likely contributing to its superior performance. These findings demonstrate important considerations in media intensification and introduce the perfusion rate reducing supplement as a viable media supplement to reduce volumetric media consumption while maintaining process performance.
Protein A chromatography is a key unit operation in biopharmaceutical purification, widely used to capture monoclonal antibodies (mAbs) from harvest cell culture fluid (HCCF). In addition to its primary function, protein A chromatography is also frequently tested for its ability to remove viruses. Although protein A specifically binds mAbs and should not interact with viruses, some studies have shown that logarithmic reduction values (LRVs) can vary between 1 and 4 log10 for retroviruses. This variance has been shown to depend on the mAb and feedstock compositions. However, it has not been evaluated so far which feedstock ingredients affect virus removal. To address this knowledge gap, a data set of virus spiking studies was analyzed and revealed a potential correlation between DNA content in HCCF and LRV of protein A chromatography. To evaluate this hypothesis, experiments with murine leukemia virus (MuLV) and retrovirus like particles (RVLP) spiked starting materials of three different mAbs were performed. In general, higher HCCF DNA levels were found to enhance LRV for both virus particles. A trend that was confirmed with RVLP spiked pure mAb. In addition, protein A chromatography washing buffers containing a detergent or a reducing agent were shown to improve LRV in RVLP spiked experiments. This study demonstrates the effect of DNA concentration on the virus removal capabilities of protein A chromatography. Findings that are especially useful when DNA is removed at early stages of the downstream process, for example by using functionalized depth filters or precipitation.
Recombinant adeno-associated virus production for gene therapy applications commonly relies on the Spodoptera frugiperda 9 (Sf9) baculovirus infection platform. The process encompasses cell expansion from flasks through intermediate-scale bioreactors to large-scale production. A critical challenge in this process is suboptimal cell proliferation during the expansion phase, which significantly impacts subsequent virus production. This study evaluates the effects of different single-use bioreactor bags on Sf9 cell growth, focusing on two potential issues: incompatibility between Sf9 cells and bag materials, and the presence of leachables and extractables from the bag. Single-use rocking motion bioreactor bag configuration, including material- and surface-related attributes, was associated with substantial differences in Sf9 cell expansion performance. While growth kinetics in shake flasks and the Pall Allegro XRS system were comparable, reduced growth rates and delayed proliferation were observed in Cytiva Cellbags. The comparable growth observed in shake flasks and the Pall XRS system, together with the lack of recovery after rocking-parameter adjustments in Cellbags, suggests that bag-associated material and surface-related factors, rather than rocking motion alone, contributed substantially to the observed growth differences. Lower bag occupancy further exacerbated growth inhibition. Washing studies showed no improvement in new bags, whereas partial recovery after initial use suggested transient bag conditioning or surface-related effects. Analytical extractables and leachables profiling was not performed; therefore, leachable-mediated contributions cannot be fully excluded. These findings highlight the importance of single-use bag selection and occupancy optimization for robust and scalable Sf9 cell expansion processes.
