A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen (DO) to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer. In this study, we investigated the effect of pH on culture performance, product titer and quality profiles by applying longitudinal multi-omics profiling, including transcriptomics, proteomics, metabolomics and glycomics, at three different culture pH set points. The subsequent systematic analysis of multi-omics data showed that pH set points differentially regulated various intracellular pathways including intracellular vesicular trafficking, cell cycle, and apoptosis, thereby resulting in differences in specific productivity, product titer and quality profiles. In addition, a time-dependent variation in mAb N-glycosylation profiles, independent of pH was identified to be mainly due to the accumulation of mAb proteins in the endoplasmic reticulum (ER) over culture time, disrupting cellular homeostasis. Overall, this multi-omics-based study provides an in-depth understanding of the intracellular processes in mAb-producing CHO cell line under varied pH conditions and could serve as a baseline for enabling the quality optimization and control of mAb production.

In vitro gut model systems permit the growth of gut microbes outside their natural habitat and are essential to the study of gut microbiota. Systems available today are limited by lack of scalability and flexibility in mode of operation. Here we describe the development of a versatile bioreactor module capable of sensing and controlling of environmental parameters such as pH control of culture medium, rate of influx and efflux of the culture medium, and aerobic/anaerobic atmosphere. Modules can be linked in series to construct a model of a digestive tract to allow the growth of microbiota in vitro. We tested the growth of a model bacterial community in a simulated mammalian gut model. The model attained and maintained a stable bacterial community that metabolized bile acids. The findings illustrate the utility of the model to grow to culture a mixed bacterial community and recapitulate biological activities such as bile acid metabolism in vitro.

Recombinant proteins are generally fused with solubility enhancer tags to improve target protein folding and solubility. However, the fusion protein strategy usually requires the use of expensive proteases to perform in vitro proteolysis and additional chromatography steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, are useful for simplifying the recombinant protein purification process, for screening molecules that fail to remain soluble after tag removal, and to promote higher yields of soluble target protein. Herein, we review controlled intracellular processing (CIP) systems, tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular protein processing regarding system design features, significant advantages and limitations of the various strategies.

Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. Further, the other byproducts of BCAA catabolism, isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures also enhance specific productivity. Additionally, the rate of production of these BCAA catabolic byproducts was negatively correlated with glucose uptake and lactate production rates. Limiting glucose supply to suppress glucose uptake and lactate production, like in case of fed-batch cultures employing HiPDOG technology, significantly enhances BCAA catabolic byproduct accumulation resulting in higher specific productivities.

Chinese Hamster Ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13C-glucose, 13C-glutamine, and 13C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition. Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-Acetyl-Glucosamine (NAG) and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.

Neural tissue engineering aims to restore function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of 3D graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p <0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly-l-lysine (PLL) was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favourable effects on the biological responses of DPSCs.

Disulfide bond reduction has been a challenging issue in antibody manufacturing, as it leads to reduced product purity, failed product specifications and more importantly, impacting drug safety and efficacy. Scientists across industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high-titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategy and development of novel approaches to achieve high product quality. Additionally, in recent years as more complex molecules emerge such as bispecific and trispecific antibodies, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative issue. Given the disulfide reduction challenges that our industry are facing, in this review, we provide a comprehensive contemporary scientific insight into the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and recovery based on our expertise in commercial and clinical manufacturing of biologics. First, this paper intended to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discussed disulfide bond reduction impact to product stability and process performance, analytical methods for detection and characterization, process control strategies and their manufacturing implementation. In addition, brief perspectives on development of future mitigation strategies will also be reviewed, including platform alignment, mitigation strategy application for bi- and tri-specific antibodies and using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from both the published papers and our internal development work.

The field of optogenetics is rapidly growing in relevance and number of developed tools. Amongst other things, the optogenetic repertoire includes light-responsive ion channels and methods for gene regulation. This review will be confined to the optogenetic control of gene expression in mammalian cells as suitable models for clinical applications. Here optogenetic gene regulation might offer an excellent method for spatially and timely regulated gene and protein expression in cell therapeutic approaches. Well-known systems for gene regulation, such as the LOV-, CRY2/CIB-, PhyB/PIF-systems, as well as other, in mammalian cells not yet fully established systems will be described. Advantages and disadvantages with regard to clinical applications are outlined in detail. Among the many unanswered questions concerning the application of optogenetics, we discuss items such as the use of exogenous chromophores and their effects on the biology of the cells and methods for a gentle, but effective gene transfection method for optogenetic tools for in vivo applications.

Exposure of Chinese hamster ovary cells (CHO) to highly concentrated feed solution during fed-batch cultivation is known to result in an unphysiological osmolality increase (>300 mOsm/kg), affecting cell physiology and morphology. Extending previous observation on osmotic adaptation, the present study investigates for the first time potential effects of hyperosmolality on CHO cells on both population and single-cell level. We intentionally exposed CHO cells to hyperosmolality of up to 545 mOsm/kg during fed-batch cultivation. Contrarily to an expected osmosis effect promoting cell shrinkage, hyperosmolality-exposed CHO cells showed a nearly triplicated volume accompanied by ablation of proliferation. On the molecular level, we observed a strong hyperosmolality-dependent increase in mitochondrial activity in CHO cells compared to control. The companion article “Hyperosmolality in CHO Culture: Effects on Proteome” provides a proteome-based insight into the effects of hyperosmolality on mitochondria. In contrast to mitochondrial activity, hyperosmolality-dependent proliferation arrest of CHO cells was not accompanied by DNA accumulation or caspase-3/7-mediated apoptosis. Notably, we demonstrate for the first time a formation of up to eight multiple, small nuclei in single hyperosmolality-stressed CHO cells. The here presented observations reveal unknown hyperosmolality-dependent morphological changes and support existing data on the osmotic response in mammalian cells.

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target specific driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is need for rapid, high-throughput, unbiased in-vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identifiy mutations that confer chemotherapeutic drug resistance.Taking the example of CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in-vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a 3D setting. Gene edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug resistant phenotype in a 3D setting, detectable by next gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug resistant cell within a large, 2500-cell, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Further, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring non-cell autonomous interactions, selection pressures, and clonal competition.

SARS-CoV-2 is an RNA coronavirus that causes severe acute pneumonia, also known as COVID 19 disease. The World Health Organization declared the COVID-19 outbreak in January 2020 and a pandemic 2 months later. Serological assays are valuable tools to study virus spread among the population and, importantly, to identify individuals that were already infected and would be potentially immune to a virus re-infection. SARS-CoV-2 Spike protein and its Receptor Binding Domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the discovery and development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this work we evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, in these antigens’ quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic ELISA tests, as denoted by the high specificity and sensitivity of the test. We have shown that, despite of the human cell host and the cell culture strategy used, for production scales ranging from 1 L to up to 30 L, final yields of approx. 2 mg and 90 mg per liter of purified bulk for Spike and RBD, respectively, could be obtained. To the best of our knowledge these are the highest yields for RBD production reported to date. An in-depth characterization of SARS CoV-2 Spike and RBD proteins was also performed, namely the antigens oligomeric state, glycosylation profiles and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS CoV 2 positive serum, for all Spike and RBD produced, and for all the storage conditions tested. Overall, we provide herein straightforward protocols to produce high-quality SARS CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-deep characterization of these proteins, correlating antigens glycosylation and biophysical attributes to performance of COVID-19 serologic tests. We strongly believe that our work will contribute to advance the current and recent knowledge on SARS-CoV-2 proteins and support the scientific society that is persistently searching for solutions for COVID-19 pandemics.

Concerns about climate change and the search for renewable energy sources together with the goal of attaining sustainable product manufacturing have boosted the use of microbial platforms to produce fuels and high-value chemicals. In this regard, Y. lipolytica has been known as a promising yeast with potentials in diverse array of biotechnological applications such as being a host for different oleochemicals, organic acid and recombinant protein production. Having a rapidly increasing number of molecular and genetic tools available, Y. lipolytica has been well studied amongst oleaginous yeasts and metabolic engineering has been used to explore its potentials. More recently, with the advancement in systems biotechnology and the implementation of mathematical modeling and high throughput omics data-driven approaches, in-depth understanding of cellular mechanisms of cell factories have been made possible resulting in enhanced rational strain design. In case of Y. lipolytica, these systems-level studies and the related cutting-edge technologies have recently been initiated which is expected to result in enabling the biotechnology sector to rationally engineer Y. lipolytica-based cell factories with favorable production metrics. In this regard, here, we highlight the current status of systems metabolic engineering research and assess the potential of this yeast for future cell factory design development.

Host cell proteins (HCPs) are process-related impurities that may co-purify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation, and often are difficult-to-purify. Why should the biopharmaceutical industry worry about these high-risk host cell proteins? What approach could be taken to understand the origin of this co-purification and to deal with these high-risk HCPs? To answer these questions, the BioPhorum Development Group (BPDG) HCP Workstream initiated a collaboration among its 26-company team with the goal of industry alignment around high-risk HCPs. A sub team was formed, in which the members performed literature searches and discussed the information available around this topic. A survey to the BPDG HCP Workstream team members led to team discussions and insights into a list of frequently seen problematic HCPs. These HCPs were further classified based on their potential impact into different risk categories that could be beneficial to the biopharmaceutical industry for targeted monitoring of those HCP impurities in CHO-produced biologics to minimize risk to product quality, safety, and efficacy.

Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, they integrate genes in semi-random patterns, and their transduction efficiency is highly variable. In this study, several non-viral gene delivery vehicles, promoters, and additional variables were compared to optimize non-viral transgene delivery and expression in both Jurkat and primary T cells. Overall, transfecting Jurkat cells in X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection efficiency (63.0±10.9% EGFP+). However, the same method yielded a much lower transfection efficiency in primary T cells (8.1±0.8% EGFP+). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans (HSPGs) detected via mRNA-sequencing. PYHIN DNA sensors (e.g., AIM2, IFI16) were also expressed at high levels in Primary T cells, which can induce apoptosis when bound to cytoplasmic DNA. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake and/or DNA sensing in the cytoplasm, so both of these factors should be considered in the development of future viral and non-viral T cell gene delivery methods.

The mechanical properties of biofilms can be used to predict biofilm deformation, for example under fluid flow. We used magnetic tweezers to spatially map the compliance of Pseudomonas aeruginosa biofilms at the micron scale, then used modeling to assess its effects on biofilm deformation. Biofilms were grown in capillary flow cells with Reynolds numbers (Re) ranging 0.28 to 13.9, bulk dissolved oxygen (DO) concentrations from 1 mg/L to 8 mg/L, and bulk calcium ion (Ca2+) concentrations of 0 and 100 mg CaCl2/L. Higher Re numbers resulted in more uniform biofilm morphologies. The biofilm was stiffer at the center of the flow cell than near the walls. Lower bulk DO led to more stratified biofilms. Higher Ca2+ led to increased stiffness and more uniform mechanical properties. Using the experimental mechanical properties, fluid-structure interaction models predicted up to 64% greater deformations for heterogeneous biofilms, compared to a homogeneous biofilms with the same average properties. However, the error depended on the biofilm morphology and flow regime. Our results show significant spatial mechanical variability exists at the micron scale, and that this variability can potentially affect biofilm deformation. The average mechanical properties, provided in many studies, should be used with caution when predicting biofilm deformation.

The heavy metals pollution represents one of the important issues in the environmental field since they are involved in many pathologies from cancer, neurodegenerative and metabolic diseases. We propose an innovative portable biosensor for the determination of traces of trivalent Arsenic (AsIII) and bivalent mercury (HgII) in water. The system implements a strategy combining two advanced sensing modules consisting in (a) a whole cell based on engineered Escherichia coli as selective sensing element towards the metals and (b) an electrochemical miniaturised silicon device with three microelectrodes and a portable reading system. The sensing mechanism relies on the selective recognition from the bacterium of given metals producing the 4-aminophenol (PAP) redox active mediator detected through a cyclic voltammetry analysis. The miniaturized biosensor is able to operate a portable, robust and high-sensitivity detection of AsIII with a sensitivity of 0.122 µA ppb-1, LoD of 1.5 ppb and a LoQ of 5 ppb. The LoD value is one order of magnitude below of the value indicated to WHO to be dangerous (10 μg/L). The system was proved to be fully versatile being effective in the detection of Hg(II) as well. A first study on Hg(II) showed sensitivity value of 2.11 µA/ppb a LOD value of 0.1 ppb and LoQ value of 0.34 ppb. Also in this case, the detected LOD was ten time lower than that indicated by WHO (1 ppb). These results pave the way for advanced sensing strategies suitable for the environmental monitoring and the public safety.

A bone regeneration scaffold is typically designed as a platform to effectively heal a bone defect while preventing soft tissue infiltration. Despite the wide variety of scaffold materials currently available, such as collagen, critical problems in achieving bone regeneration remain, including a rapid absorption period and low tensile strength as well as high costs. Inspired by extracellular matrix protein and topographical cues, we developed a polycaprolactone-based scaffold for bone regeneration using a soluble eggshell membrane protein (SEP) coating and a nanotopography structure for enhancing the physical properties and bioactivity. The scaffold exhibited adequate flexibility and mechanical strength as a biomedical platform for bone regeneration. The highly aligned nanostructures and SEP coating were found to regulate and enhance cell morphology, adhesion, proliferation, and differentiation in vitro. In a calvarial bone defect mouse model, the scaffolds coated with SEP applied to the defect site promoted bone regeneration along the direction of the nanotopography in vivo. These findings demonstrate that bone-inspired nanostructures and SEP coatings have high potential to be applicable in the design and manipulation of scaffolds for bone regeneration.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy (“self-eating”) is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human IgG1. Interestingly, the addition of 1 to 4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum 2-fold increase in volumetric productivity was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.