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.

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.

Regeneron's bispecific technology, based on a fully human IgG antibody construct, consists of two distinct heavy chains and a common light chain co-expressed in a single cell. The co-expression of the two distinct heavy chains, one of which contains a dipeptide substitution that ablates Protein A binding, leads to the formation of three species with varying degrees of avidity to Protein A: the heterodimer bispecific and two parental homodimer impurities. The avidity difference between the three species can be leveraged for both bulk capture of the bispecific and resolution of the bispecific from the homodimer impurities via a ternary resolving Protein A chromatographic separation on commercially available resins amenable to large scale Good Manufacturing Practice (GMP) production. This case study highlights the efficiency of a mechanistic modeling approach for the optimization and characterization of ternary resolving Protein A unit operations. A mechanistic model, comprised of a general rate transport model and colloidal particle adsorption (CPA) affinity isotherm, was developed to describe this ternary separation for a model bispecific. The model was calibrated and validated with 10 laboratory experiments and demonstrated an ability to predict bispecific yield within 3% and bispecific purity within 1% on average. The model was then used to optimize the process and characterize the design space, while providing mechanistic insight into the process.

The deployment of advanced modeling and machine learning in biopharmaceutical process control has been limited by legacy fragmented automation infrastructure and a lack of model lifecycle governance. We present BioOps, a modular automation framework for biopharmaceutical advanced process control (APC). The framework incorporates established Machine Learning Operations (MLOps) principles, including version-controlled model registries, containerized deployment, and continuous integration/continuous delivery (CI/CD) pipelines. BioOps explicitly decouples model development from model execution within the control architecture. This decoupling enables process scientists to deploy, test, and iteratively refine mathematical models and control strategies in a flexible manner. These workflows can be applied across heterogeneous bioreactor platforms and are executed entirely in-house, without reliance on vendor-specific solutions or custom system integration. In addition, BioOps embeds traceability, auditability, and model lifecycle management directly into the control stack. As a result, the framework aligns with regulatory expectations while continuing to support agile, in-house experimentation and development. Case studies across a geographically distributed set of bioreactors demonstrate BioOps' versatility in PAT-driven feedback control, hybrid model-based nutrient and glucose feeding, adaptive phase transitions, and cross-scale deployment. By operationalizing model-centric control through an MLOps-inspired architecture, BioOps provides a practical foundation for scalable, reproducible, and future-ready biomanufacturing.

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.

Haptoglobin (Hp) is a polymorphic acute phase α-2 glycoprotein found in plasma that plays a critical role in binding, neutralizing, and removing cell-free hemoglobin (Hb) from the circulation. Under clinical conditions characterized by high levels of hemolysis, such as in patients with sickle cell disease (SCD), large quantities of cell-free Hb are released from lysed red blood cells (RBCs) into the circulation and bind to Hp. This interaction reduces the plasma Hp concentration below basal levels and diminishes its Hb-binding capacity. Therefore, plasma-derived Hp has the potential to be used therapeutically to scavenge, neutralize, and remove excess cell-free Hb from the blood, thus preventing Hb-mediated toxicity. This provides strong motivation to purify Hp at high purity using sustainable sources, such as waste plasma fractions from the Cohn plasma fractionation process used to produce human serum albumin from pooled plasma. Starting from human Cohn fraction IV, we first enriched an Hp-rich fraction using tangential flow filtration (TFF), which was then used as the starting material for purification. Hp was subsequently purified from this fraction using hydrophobic interaction chromatography (HIC) to homogeneity. The final Hp purity reached 98% by SDS-PAGE densitometry and 87% by trypsin digest LC-MS/MS analysis. The purified Hp was further characterized to determine its molecular weight, secondary structure, Hb-binding capacity, and binding kinetics using biophysical techniques including MALDI-TOF, circular dichroism, size exclusion HPLC, and UV-visible stopped-flow spectroscopy. More importantly, the simplicity and efficiency of the TFF-HIC workflow demonstrated strong potential for scalable Hp production.

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.

Chronic myeloid leukemia (CML), one type of blood cancer, is a clonal myeloproliferative disorder of hematopoietic stem cells that originated from bone marrow (BM). Imatinib (IM) resistance in CML treatment has been frequently reported, which mainly results from the complex microenvironment of BM. BM is a viscous tissue where cells are mechanically responsive to both the stiffness of matrices and the viscosity of surrounding fluid. Although the effect of stiffness on CML drug resistance has been revealed, the role of BM viscosity on IM resistance in CML is unclear. In this study, a BM-like viscous culture medium was prepared to investigate the effect of microenvironmental viscosity on IM resistance in CML cells. The culture media with viscosity ranging from 47.6 to 634.9 cP were prepared to establish viscosity-controlled culture media covering the reported physiological range of bone marrow and extending into an exploratory higher-viscosity condition. CML cells were suspended and cultured in the viscous culture media added or without addition of IM for 48 or 72 h. Cell viability decreased with IM concentration but increased with microenvironmental viscosity. The results suggested that increased microenvironmental viscosity reduced the sensitivity of CML cells to IM. The possible mechanism might involve the upregulated expression of IM resistance-related genes, reduced apoptosis rates, and altered expression patterns of the BCR::ABL1 gene.

Lipid droplets (LDs) are dynamic organelles that serve as metabolic hubs and emerging regulators of immune cell fate. Although LDs have been implicated in immune regulation, how LD metabolism is remodeled during neutrophil differentiation and how stage-specific LD dynamics shape mature neutrophil function remain poorly defined. Here, we profiled LD dynamics during neutrophil development and evaluated their role in innate immune function. Using stimulated Raman scattering (SRS) microscopy, we performed label-free, quantitative mapping of LD accumulation in two complementary differentiation systems: murine Hoxb8 myeloid progenitors and human pluripotent stem cell (hPSC)-derived neutrophils. To define metabolic requirements, we pharmacologically modulated LD biosynthesis and catabolism throughout neutrophil differentiation. Perturbing LD metabolism did not impair differentiation efficiency or lineage commitment, but significantly altered the functional output of mature neutrophils. In the murine system, inhibition of adipose triglyceride lipase (ATGL)-mediated LD breakdown enhanced reactive oxygen species (ROS) production and increased anti-tumor cytotoxicity against GL261 glioma cells. In the hPSC model, ATGL inhibition during the myeloid progenitor-to-neutrophil transition selectively increased intracellular LD accumulation without compromising neutrophil yield or purity. This metabolic rewiring also elevated ROS production in hPSC-derived neutrophils, although cytotoxic enhancement against U87MG glioblastoma cells was less pronounced than in the murine system. Collectively, these findings define a stage-specific LD metabolic landscape during neutrophil development and highlight targeted LD modulation as a potential strategy to enhance the functional potency of therapeutic neutrophils.

The production of Fab therapeutics is frequently challenged by an imbalanced expression of light chains (LC) and heavy chains (HC), often resulting in LC-associated impurities. In this work, we evaluated multimodal chromatography as an alternative to affinity chromatography for the removal of an LC impurity. A panel of multimodal CEX and multimodal AEX resins was screened using linear salt and pH gradients to assess Fab monomer and LC impurity selectivities. Product and impurity characterization was carried out using analytical size-exclusion chromatography. Screening data provided insight into the relative contributions of hydrophobic and electrostatic interactions in the Fab and LC impurity selectivities across different multimodal ligands. A previously developed separability scoring metric (Fractional Peak Overlap) was employed to identify Eshmuno HCX and CMM HyperCel as the top resin candidates from the screening data. Finally, bench-scale refinement was carried out to develop an Eshmuno HCX bind-elute step with a 95%-100% recovery and 95%-98% purity of Fab monomer and a CMM HyperCel bind-elute step with a 98%-99% recovery and 98% purity of Fab monomer. This work demonstrates the efficacy of this PD workflow and the utility of multimodal chromatography for removal of LC impurities from Fabs.

Low-pH viral inactivation (LPVI) is a widely used, validated unit operation for enveloped virus clearance in antibody manufacturing. However, acid exposure during Protein A (ProA) elution and subsequent low-pH holds can destabilize some antibodies and Fc-containing modalities, increasing aggregation risk and product loss-a challenge that is particularly acute for multi-specific antibody formats and other pH-labile Fc-containing modalities. Mild-pH ProA capture strategies protect such molecules, but the resulting higher-pH eluates often require re-acidification to meet conventional LPVI setpoints, reintroducing low-pH stress. Here we evaluate a dual-mechanism strategy in which mild acidity is combined with low concentrations of non-ionic detergent to accelerate inactivation without prolonged exposure to strongly acidic conditions. Using xenotropic murine leukemia virus (X-MuLV) spiked into ProA eluates, 0.10% N-methylglucamide (Mega-10) at pH 3.91 reduced infectivity to below the assay limit of detection within 2.5 min (log10 reduction factor [LRF] ≥ 5.40), whereas low pH alone or detergent alone produced substantially lower reductions over the same interval. Similarly, 0.15% Mega-10 at pH 4.05 achieved nondetectable infectivity within 10 min (LRF ≥ 5.81). Comparable time compression was observed with Tween 80 or Tween 20 at pH ~3.97, reducing time-to-nondetectable infectivity from 120 min (low pH alone) to 5 min. The operating window was tunable through paired adjustment of detergent concentration and pH, and feasibility was demonstrated at pH 4.56 with low detergent levels, offering a practical solution for pH-labile products and higher-pH eluates.

Continuous chromatography for Protein A capture of monoclonal antibodies (mAbs) and related biotherapeutics is gaining popularity for higher throughput and reduced costs but introduces challenges for development and modeling. We present a streamlined, single-breakthrough-curve approach that predicts maximum and operating binding capacities (MBC and OBC, respectively) for continuous Protein A chromatography of monoclonal antibodies and derivatives. Our empirically-calibrated model-intended for early-stage, material-limited process development-enables accurate OBC prediction (root mean squared error [RMSE]≤ 5% for four distinct proteins) using less than 1 g of protein. This approach reduces the experimental timeline by ~2 weeks and is best suited for rapid screening and initial process optimization, not full design-space determination. Results confirm the model's accuracy across molecules, with clear practical advantages for accelerating preclinical pipeline advancement.

Perfusion bioreactor systems enable intensified bioprocessing with high cell densities and volumetric productivities in monoclonal antibody (mAb) manufacturing. However, their widespread adoption remains constrained by the high consumption of chemically defined cell culture media. In this study, we introduce a multi-stage electrokinetic waste separation process that regenerates spent media, recovering up to 87.5% of the spent-medium volume for reuse. Using a mock-perfusion model, the regeneration-recycle processes were shown to sustain high-density Chinese hamster ovary cell growth, compared with spent media recycling without regeneration. Integration into a self-recycling perfusion bioreactor (350 mL working volume) operating with 75% regenerated media sustained the target viable cell density and antibody titer, with limited reductions to cell-specific growth rate (~31%) and cell-specific productivity (~9%), supporting the feasibility of high-ratio media recycling. Techno-economic modeling of large-scale scenarios revealed that media regeneration could offer a path to $15/g COGs, thereby increasing the accessibility of mAbs. Techno-economic modeling of hypothetical large-scale manufacturing scenarios indicated that the experimentally demonstrated process could reduce cost of goods (COG) by approximately 9%, and identified a theoretical pathway toward ~$15/g COG contingent upon multi-cycle regeneration processes that could be developed in the future. These results support regenerative perfusion as a promising process-intensification strategy that, with further development, may be adaptable to other mammalian cell culture platforms where media cost and sustainability are critical bottlenecks.

Reliable assessment of cellular state remains a challenge in cell therapy manufacturing and regenerative medicine. Proliferative capacity, senescence, and metabolic activity are traditionally measured using labor-intensive assays, limiting timely and integrated assessment of cellular state. Here, we apply attenuated total reflection-Fourier transform infrared spectroscopy coupled with multivariate analysis to assess whether intrinsic vibrational fingerprints of mesenchymal stem cells reflect coordinated functional changes during in vitro expansion. By integrating proliferation kinetics, metabolite fluxes, and senescence-associated phenotype into a composite functional score, we mapped progressive functional decline across passages onto infrared spectral changes. Spectral alterations were associated with shifts in lipid-related and protein-associated regions as senescence accumulated. Despite strong donor-specific differences, we identified conserved spectral fingerprint regions that tracked functional decline across donor samples analyzed. These shared biochemical signatures were associated with the prediction of senescence progression and the estimation of functional passage in an independent donor sample. Together, our results show that intrinsic vibrational fingerprints capture coordinated biochemical changes during cellular decline and provide a label-free, rapid means to assess cellular state.