Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). An acellular collagen platform with an endothelial blood vessel served as control. SUM149 and MDA-MB-231 platforms exhibited a significantly (p<0.05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of VEGF (p<0.05) and increased collagen ECM porosity compared to non-IBC MDA-MB-231 (p<0.05) and control (p<0.01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughout tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.
The challenge of introducing new technologies into established industries is not a problem unique to the biopharmaceutical industry. However, it may be critical to the long-term competitiveness of individual manufacturers and, more importantly, the ability to deliver therapies to patients. This is especially true for new treatment modalities including cell and gene therapies. We review several barriers to technology adoption which have been identified in various public forums including business, regulatory, technology, and people-driven concerns. We also summarize suitable enablers addressing one or more of these barriers along with some suggestions for developing additional synergies.
Thermobifida fusca cutinase ( TfC ) is a carboxylesterase (CE) that degrades the environmental pollutant, polyethylene terephthalate (PET). TfC also acts upon PET’s degradation intermediates (DIs), such as oligoethylene terephthalate (OET), and bis-/mono-hydroxyethyl terephthalate (BHET/MHET), to convert these into terephthalic acid (TPA), the terminal product of PET degradation. We examined TfC’s surface, compared it to that of other CEs, and performed molecular docking and MD simulations with an OET, 2HE-(MHET) 3, to understand interactions between TfC’s surface and the OET, at TfC’s active site as well as vicinal regions. We mutated 17 residues on TfC’s surface, mostly individually, but sometimes using pairs of mutations, to see how these modulate TfC’s activity. Most mutants/variants showed a decrease in activity against solid PET. Some killed activity completely. However, three mutations (L90F, F209I and F249R), made using a background mutation (G62A) already reported to improve activity by almost ~2.0-fold, yielded increases in activity that were between ~1.3- and ~2.0-fold higher than that of G62A TfC (which we found to display a ~1.7-fold increase in activity over TfC, in our own experiments). TfC variants, G62A/F249R, and G62A/F209I, exhibit the highest activities yet observed in any TfC mutants/variants, against PET, and BHET, respectively.
Cancer is one of the major health-related issues affecting the population worldwide and subsequently accounts for the second-largest death. Genetic and epigenetic modifications in oncogenes or tumor suppressor genes affect the regulatory systems that lead to the initiation and progression of cancer. Conventional methods, including chemotherapy/radiotherapy/appropriate combinational therapy and surgery, are being widely used for theranostics of cancer patients. Surgery is useful in treating localized tumors, but it is ineffective in treating metastatic tumors, which spread to other organs and result in a high recurrence rate and death. Also, the therapeutic application of free drugs is related to substantial issues such as poor absorption, solubility, bioavailability, high degradation rate, short shelf-life, and low therapeutic index. Therefore, these issues can be sorted out using nano lipid-based carriers (NLBCs) as promising drug delivery carriers. Still, at most, they fail to achieve site targeted drug delivery and detection. This can be achieved by selecting a specific ligand/antibody for its cognate receptor molecule expressed on the cancer cell surface. In this review, we have mainly discussed the various types of ligands used to decorate NLBCs. A list of the ligands used to design nanocarriers to target malignant cells specifically has been extensively undertaken, and the approved ligand decorated lipid-based nanomedicines with their clinical status has been explained in tabulated form to provide a wider scope to the readers regarding ligand coupled NLBCs.
Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.
Chemically defined (CD) media are routinely used in the production of biologics in Chinese Hamster Ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multi-factorial and experimental design of experiment (DoE) approaches or complex mathematical models of cellular metabolism or gene expression systems. Moreover, the majority of these efforts are aimed at amino acids since they constitute essential nutrients in CD media as they directly contribute to biomass and protein production. In this study, we demonstrate the utilization of multi-variate data analytics (MVDA) coupled with amino acid stoichiometric balances (SBs) to increased cell growth and mAb productivity in efforts to reduce CD media development efforts. SBs measure the difference between theoretical demand of amino acids and the empirically measured fluxes to identify metabolic states of the cell. When coupled with MVDA, the statistical models were not only able to highlight key amino acids towards cell growth or productivity, but also provided direction on metabolic favorability of the amino acid. Experimental validation of our approach resulted in a 55% increase in total cell growth and about an 80% increase in total mAb productivity. Increased specific consumption of stoichiometrically balanced amino acids and decreased specific consumption of glucose was also observed in optimized CD media suggesting favorable consumption of desired nutrients and a potential for energy redistribution towards increased cellular growth or mAb productivity.
Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the E. coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with non-natural amino acids incorporated at specific locations for producing homogeneous antibody drug conjugates. While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for non-natural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a five-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing non-natural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.
Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this work, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.
Adoptive cell immunotherapy with chimeric antigen receptor (CAR) T cell has brought a revolutionary means of treatment for aggressive diseases such as hematologic malignancies and solid tumors. Over the last decade, FDA approved three types of CAR-T cells against CD19 hematologic malignancies, including Tisagenlecleucel (Kymriah), Axicabtagene ciloleucel (Yescarta), and Brexucabtagene autoleucel (Tecartus). Despite outstanding results gained from different clinical trials, CAR-T cell therapy is not free from side effects and toxicities, and needs careful investigations and improvements. Gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system has emerged as a promising tool to address some of the CAR-T therapy hurdles. Using CRISPR/Cas9 technology, CAR expression as well as other cellular pathways can be modified in various ways to enhance CAR-T cell’s anti-tumor function and persistence in immunosuppressive tumor microenvironment. CRISPR/Cas9 technology can also be utilized to reduce CAR-T cells toxicity and side effects. Hereby, we discuss the practical challenges and hurdles related to the accuracy, efficiency, efficacy, safety and delivery of CRISPR/Cas9 technology to the genetically engineered-T cells. Combining of these two state-of-the-art technologies, CRISPR/Cas9 and CAR-T cells, the field of oncology has an extraordinary opportunity to enter a new era of immunotherapy, which offers novel therapeutic options for different types of tumors.
Seasonal influenza infection waves occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight-forward. Nevertheless, compared to the recent Covid-19 pandemic where a vaccine is not yet available, influenza vaccine manufacturing would be a major bottleneck for the rapid supply of billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this work, we present an integrated process for the production of inactivated influenza A virus vaccines based on a MDCK suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10(HAU/100 µL) were achieved using fast growing MDCK cells at concentrations up to 9.5 × 106 cells/mL infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of two membrane-based chromatography steps enabled full recovery for the virus capture and up to 80 % recovery for the virus polishing step, respectively. Purified virus particles showed a homogenous size distribution around a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (SRID assay), the level of total protein was 58 µg and the level of host cell DNA contamination was below 10 ng. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4 to 5 days makes this process competitive not only to other cell-based processes, but to egg-based processes as well.
The serious drawbacks of conventional pancreatic ductal adenocarcinoma (PDAC) therapy like nonspecific toxicity and high resistance to chemo and radiation therapy, prompted the development and application of countless siRNA-based therapeutics. Significant technological success has been achieved in this area; however, the major challenges to siRNA-based therapeutics becoming a new paradigm in the pancreatic cancer therapy stem from enzymatic digestion, off-target effects, difficulty to enter cells, induction of innate immune responses, and renal clearance. Recent advances in drug delivery systems hold great promise for improving siRNA-based therapeutics and developing a new class of drugs, nano-siRNA drugs. However, a number of fundamental questions, regarding toxicity, immunostimulation, and poor knowledge of nano-bio interactions, need to be addressed before clinical translation. In this review, we provide recent achievements in designing and development of various non-viral delivery vehicles for pancreatic cancer therapy. More importantly, co-delivery of conventional anticancer drugs with siRNA as a new revolutionary pancreatic cancer combinational therapy is completely discussed.
Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Further, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, i.e., isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for KS (9.3-fold higher) and kcat (4.8-fold lower) compared to corresponding KM and kcat values of isolated CHMO, but a favorable KI for cyclohexanone (5.3-fold higher). The unfavorable KS and kcat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, in order to exploit the high potential of biofilms regarding process stability. Suspended cells showed an only 1.8-fold higher KS, but 1.3- and 4.2-fold higher kcat and KI values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.
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.
Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low-cost production, although low yields remain the major challenge. The studies on scaling-up to pilot-scale bioreactors, especially in conventional stainless-steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled-up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically-regulated transgenic rice cell suspension cultures in a 40-L pilot-scale STB. Employing cyclical operation together with a simplified-process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab-scale (5-L) bioreactor studies, we found consistent maximum total active rrBChE production level of 46-58 µg/g fresh weight in four cycles over 82 days of continuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot-scale STB to be equivalent to the lab-scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L-1 day-1, respectively, which are comparable to the lab-scale culture. Here, we demonstrate pilot scale bioreactor performance using a metabolically-regulated transgenic rice cell culture for long-term, reproducible, and sustained production of rrBChE.
Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle--dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses of adherent CHO K1 cultures, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle--dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. Indeed, the mitochondrial genome sequencing method developed here enables the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.
As issues surrounding depleting fossil fuels, climate change, and various other environmental impacts are becoming more prevalent, there is a growing interest in technological shifts toward a bio-based economy. Various advanced biotechnological tools have been developed to customize cell factories for the production of a wide range of complex fine chemicals from renewable feedstock. Herein, we report development of a microbial bioprocess for high-level and potentially economical production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis solely from glycerol (~30 g l-1) under both microaerobic and aerobic conditions, achieving up to 5.95 g l-1 (36.9% yield) and 6.93 g l-1 (50.9% yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.
Vaccines provide effective protection against many infectious diseases as well as therapeutics for some serious diseases, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, that was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a higher doubling time in both 2D and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.
Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms due to the ability to produce complex biologics, use of simple chemically-defined, animal component-free media, robustness of host cells, and biosafety. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous techno-economic models or analyses have been published. Here we present techno-economic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.