Proteolytic degradation of recombinant proteins in Chinese hamster ovary (CHO) cells remains a major challenge in biopharmaceutical manufacturing, often reducing product yield and quality. Matriptase-1, a type II transmembrane serine protease, has been identified as a key contributor to unwanted proteolysis. This study investigates hepatocyte growth factor activator inhibitor-1 (HAI-1) overexpression as a strategy to mitigate matriptase-1-mediated degradation in CHO cell culture. Using an IL-12 IgG1 Fc fusion protein and a multi-specific antibody (M molecule) as model proteins, we employed genetic and biochemical approaches to assess the impact of Chinese hamster HAI-1 overexpression on protein quality and yield. Our results demonstrate that HAI-1 overexpression effectively inhibits matriptase-1 activity, achieving up to a 98% reduction in proteolytic clipping while maintaining cell growth, viability, and product quality. Compared to other protease control strategies, HAI-1 overexpression presents a practical and scalable solution that does not disrupt essential cellular functions. These findings establish HAI-1 as a key modulatory tool in CHO-based protein production, with implications for reducing proteolysis of therapeutic proteins and optimizing biopharmaceutical manufacturing processes.

The cover image is based on the Research Article Contributions of Chinese hamster ovary cell derived extracellular vesicles and other cellular materials to hollow fiber filter fouling during perfusion manufacturing of monoclonal antibodies by Yixiao Zhang et al., https://doi.org/10.1002/bit.28674.

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.

Recent studies have unveiled the unique roles of extracellular vesicles (EVs) in various cellular processes including protein degradation, transport, and intercellular communication. However, the EVs of Chinese hamster ovary (CHO) cells, the workhorse of biologics manufacturing, have not been well-characterized despite their significant roles in protein production. Herein, we successfully isolated CHO EVs from CHO fed-batch cultures and identified their messenger RNA (mRNA) and micro RNA (miRNA) contents through next-generation sequencing. We found that mRNAs corresponding to oxidative phosphorylation were highly enriched in microvesicles (large EVs) but absent in exosomes (small EVs). We also found that both large EVs and small EVs had enriched mRNA species corresponding to key signaling pathways for cell proliferation, survival, and growth, including the TGFβ and PI3K/Akt pathways. In addition, the enrichment of miR-196a-5p in both small EVs and large EVs suggests an anti-apoptotic and proliferative function for EVs through intercellular communication. The identification of these mRNAs and miRNAs associated with cell growth and survival sheds light on the potential role of extracellular vesicles in the context of biologics manufacturing and may help further optimize CHO biologics production.

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 has higher mechanical tension in regions of cell adhesion, on and in response to nanotopographical cues. In addition, we found that substrate stiffness also regulated the mechanical tension of syndecan-1 and altered its binding to actin, myosin iiB and signaling intermediates including Src, PKA and FAK. Application of fluidic shear stress created a gradient in tension in syndecan-1 and led to enhanced association with actin, Src, myosin IIb and other cytoskeleton related molecules. Overall, our studies support that syndecan-1 is responsive to the mechanical environment of the cells and alters its association with actin and signaling intermediates in response to mechanical stimuli.

α-Synuclein overexpression and aggregation are linked to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and several other neurodegenerative disorders. In addition to effects in the cell body, α-synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess α-synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. We show here that acute introduction of excess human α-synuclein at a classic vertebrate synapse, the lamprey reticulospinal (RS) synapse, selectively impaired the uncoating of clathrin-coated vesicles (CCVs) during synaptic vesicle recycling, leading to an increase in endocytic intermediates and a severe depletion of synaptic vesicles. Furthermore, human α-synuclein and lamprey γ-synuclein both interact in vitro with Hsc70, the chaperone protein that uncoats CCVs at synapses. After introducing excess α-synuclein, Hsc70 availability was reduced at stimulated synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic vesicle trafficking defects. In support of this hypothesis, increasing the levels of exogenous Hsc70 along with α-synuclein ameliorated the CCV uncoating and vesicle recycling defects. These experiments identify a reduction in Hsc70 availability at synapses, and consequently its function, as the mechanism by which α-synuclein induces synaptic vesicle recycling defects. To our knowledge, this is the first report of a viable chaperone-based strategy for reversing the synaptic vesicle trafficking defects associated with excess α-synuclein, which may be of value for improving synaptic function in PD and other synuclein-linked diseases.

Receptor internalization by endocytosis regulates diverse cellular processes from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein binding interactions. However, recent work suggests that biophysical factors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here we show that receptor uptake is well-described by a thermodynamic tradeoff between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. In agreement with a simple thermodynamic model, all data follow a single trend relating occupancy of the endocytic structure to occupancy of the surrounding plasma membrane. This fundamental scaling law provides a simple tool for predicting the impact of biophysical factors on receptor uptake rate, including receptor size, affinity for endocytic structures, and expression level. In agreement with these predictions, our results demonstrate how receptors of varying size and affinity compete for limited space within crowded endocytic structures. Understanding this biophysical competition is important for predicting receptor uptake in settings where endocytosis is saturated by receptor overexpression, as is the case in many tumor cells. More broadly, this work demonstrates that bulky ligands can drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

Nanoparticle-based drug delivery systems have the potential to target diseased cells on the basis of their receptor expression profiles. Toward this goal, synthetic materials conjugated with antibodies against specific cellular receptors have been shown to concentrate in solid tumors. However, chemical conjugation reactions are difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. To harness cellular capabilities, here we demonstrate that plasma membrane vesicles (PMVs) derived from engineered donor cells can display multifunctional transmembrane proteins that precisely target cells on the basis of the expression level of a specific cellular receptor. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs displaying protein ligands with affinity for epidermal growth factor receptor (EGFR) bound at increasing concentrations to breast cancer cells that expressed increasing levels of EGFR. As an example of the generality of this approach, we developed PMVs displaying a single-domain antibody against green fluorescence protein (eGFP), which bound to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. To further demonstrate the versatility of PMVs, we engineered targeting proteins with serial repeats of the ligand domain to mimic the high-affinity binding mechanisms commonly observed in multivalent biochemical interactions. Collectively, these results demonstrate the flexibility and high selectivity of PMVs as a novel biomaterial for cell targeting. Moving forward, the use of engineered donor cells to produce PMVs will enable the development of true multi-functional materials with diverse applications from drug delivery to tissue engineering.

Transport of biomolecules, drugs, and other reagents across the cell's plasma membrane barrier is an inefficient and poorly controlled process, despite its fundamental importance to biotechnology, cell biology, and pharmaceutics. In particular, insufficient membrane permeability frequently limits the accumulation of drugs and reagents in the cytoplasm, undermining their efficacy. While encapsulating drugs in particles increases uptake by cells, inefficient release of drugs from these particles into the cytoplasm ultimately limits drug efficacy. In contrast, gap junctions provide a direct route to the cytoplasm that bypasses the plasma membrane. As transmembrane channels that physically connect the cytoplasm of adjacent cells, gap junctions permit transport of a diverse range of molecules, from ions and metabolites to siRNA, peptides, and chemotherapeutics. To utilize gap junctions for molecular delivery we have developed Connectosomes, cell-derived lipid vesicles that contain functional gap junction channels and encapsulate molecular cargos. Here we show that these vesicles form gap junction channels with cells, opening a direct and efficient route for the delivery of molecular cargo to the cellular cytoplasm. Specifically, we demonstrate that using gap junctions to deliver the chemotherapeutic doxorubicin reduces the therapeutically effective dose of the drug by more than an order of magnitude. Delivering drugs through gap junctions has the potential to boost the effectiveness of existing drugs such as chemotherapeutics, while simultaneously enabling the delivery of membrane-impermeable drugs and reagents.

Receptor internalization by endocytosis controls diverse cellular processes from the rate of nutrient uptake to the timescale of essential signaling events. While many of the biochemical motifs that enable uptake have been thoroughly investigated, the physical factors that influence receptor internalization remain poorly understood. Specifically, recent work from several groups has demonstrated that the steric bulk of receptors inhibits their uptake by multiple types of trafficking vesicles including caveolae, COPII vesicles, and clathrin coated pits. However, the mechanism by which endocytic structures differentiate among receptors of different sizes to control their uptake remains unclear. Using bulky ligands to acutely increase receptor size, here we show that receptor uptake is governed by a thermodynamic balance between receptor-vesicle binding energy and the entropic cost of confining receptors within nascent vesicles. Specifically, we used quantitative fluorescence imaging to precisely map the distribution of receptors among clathrin-coated pits at the plasma membrane surface. In agreement with a simple thermodynamic model, these data show that the increased entropic cost of internalizing bulky ligands inhibits receptor uptake, driving accumulation of ligated receptors at the plasma membrane. Further, by examining the differential uptake of two receptors competing for space within the same population of endocytic structures, our results reveal the relative importance of receptor size and receptor affinity for endocytic sites. From a biological perspective, these results can predict how processes such as ligand binding and dimerization, which alter both the effective size of receptors and their biochemical affinity for endocytic structures, will impact receptor uptake rate. From an applied perspective, this work guides the design of bulky ligands that can drive the accumulation of specific receptors at the plasma membrane surface, a tool that may be useful for modulating diverse pathways in cell physiology and signaling.

Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury.

Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering.

Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this “speed limit” could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.