Stem cell biomanufacturing under uncertainty: A case study in optimizing red blood cell production.
ABSTRACT: As breakthrough cellular therapy discoveries are translated into reliable, commercializable applications, effective stem cell biomanufacturing requires systematically developing and optimizing bioprocess design and operation. This article proposes a rigorous computational framework for stem cell biomanufacturing under uncertainty. Our mathematical tool kit incorporates: high-fidelity modeling, single variate and multivariate sensitivity analysis, global topological superstructure optimization, and robust optimization. The advantages of the proposed bioprocess optimization framework using, as a case study, a dual hollow fiber bioreactor producing red blood cells from progenitor cells were quantitatively demonstrated. The optimization phase reduces the cost by a factor of 4, and the price of insuring process performance against uncertainty is approximately 15% over the nominal optimal solution. Mathematical modeling and optimization can guide decision making; the possible commercial impact of this cellular therapy using the disruptive technology paradigm was quantitatively evaluated.
Project description:Collagen is a natural polymer found abundantly in the extracellular matrix (ECM). It is easily extracted from a variety of sources and exhibits excellent biological properties such as biocompatibility and weak antigenicity. Additionally, different processes allow control of physical and chemical properties such as mechanical stiffness, viscosity and biodegradability. Moreover, various additive biomanufacturing technology has enabled layer-by-layer construction of complex structures to support biological function. Additive biomanufacturing has expanded the use of collagen biomaterial in various regenerative medicine and disease modelling application (e.g., skin, bone and cornea). Currently, regulatory hurdles in translating collagen biomaterials still remain. Additive biomanufacturing may help to overcome such hurdles commercializing collagen biomaterials and fulfill its potential for biomedicine.
Project description:The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.
Project description:Colchicine is one of the oldest plant-based medicines used to treat gout and one of the most important alkaloid-based antimitotic drugs with anticancer potential, which is commercially extracted from Gloriosa superba. Clinical trials suggest that colchicine medication could prevent atrial fibrillation recurrence after cardiac surgery. In addition, therapeutic colchicine is undergoing clinical trials to treat non-diabetic metabolic syndrome and diabetic nephropathy. However, the industrial-scale biomanufacturing of colchicine have not yet been established. Clearly, further studies on detailed biorhizome-specific transcriptome analysis, gene expression, and candidate gene validation are required before uncover the mechanism of colchicine biosynthesis and biorhizome-based colchicine biomanufacturing. Annotation of 32312 assembled multiple-tissues transcripts of G. superba represented 15088 unigenes in known plant specific gene ontology. This could help understanding colchicine biosynthesis in G. superba. This review highlights the biorhizomes, rhizome specific genes or gene what expressed with high level in rhizomes, and deep fluid dynamics in a bioreactor specifically for the biomanufacture of colchicine.
Project description:The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ?20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ?10(9)particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use.
Project description:Especially in biomanufacturing, methods to design optimal experiments are a valuable technique to fully exploit the potential of the emerging technical possibilities that are driving experimental miniaturization and parallelization. The general objective is to reduce the experimental effort while maximizing the information content of an experiment, speeding up knowledge gain in R&D. The approach of model-based design of experiments (known as MBDoE) utilizes the information of an underlying mathematical model describing the system of interest. A common method to predict the accuracy of the parameter estimates uses the Fisher information matrix to approximate the 90% confidence intervals of the estimates. However, for highly non-linear models, this method might lead to wrong conclusions. In such cases, Monte Carlo sampling gives a more accurate insight into the parameter's estimate probability distribution and should be exploited to assess the reliability of the approximations made through the Fisher information matrix. We first introduce the model-based optimal experimental design for parameter estimation including parameter identification and validation by means of a simple non-linear Michaelis-Menten kinetic and show why Monte Carlo simulations give a more accurate depiction of the parameter uncertainty. Secondly, we propose a very robust and simple method to find optimal experimental designs using Monte Carlo simulations. Although computational expensive, the method is easy to implement and parallelize. This article focuses on practical examples of bioprocess engineering but is generally applicable in other fields.
Project description:The modern cell-free protein synthesis (CFPS) system is expanding the opportunity of cell-free biomanufacturing as a versatile platform for synthesizing various therapeutic proteins. However, synthesizing human protein in the bacterial CFPS system remains challenging due to the low expression level, protein misfolding, inactivity, and more. These challenges limit the use of a bacterial CFPS system for human therapeutic protein synthesis. In this study, we demonstrated the improved performance of a customized CFPS platform for human therapeutic protein production by investigating the factors that limit cell-free transcription-translation. The improvement of the CFPS platform has been made in three ways. First, the cell extract was prepared from the rare tRNA expressed host strain, and CFPS was performed with a codon-optimized gene for Escherichia coli codon usage bias. The soluble protein yield was 15.2 times greater with the rare tRNA overexpressing host strain as cell extract and codon-optimized gene in the CFPS system. Next, we identify and prioritize the critical biomanufacturing factors for highly active crude cell lysate for human protein synthesis. Lastly, we engineer the CFPS reaction conditions to enhance protein yield. In this model, the therapeutic protein filaggrin expression was significantly improved by up to 23-fold, presenting 28 ± 5 ?M of soluble protein yield. The customized CFPS system for filaggrin biomanufacturing described here demonstrates the potential of the CFPS system to be adapted for studying therapeutic proteins.
Project description:Engineering organ-specific tissues for therapeutic applications is a grand challenge, requiring the fabrication and maintenance of densely cellular constructs composed of ~108 cells/ml. Organ building blocks (OBBs) composed of patient-specific-induced pluripotent stem cell-derived organoids offer a pathway to achieving tissues with the requisite cellular density, microarchitecture, and function. However, to date, scant attention has been devoted to their assembly into 3D tissue constructs. Here, we report a biomanufacturing method for assembling hundreds of thousands of these OBBs into living matrices with high cellular density into which perfusable vascular channels are introduced via embedded three-dimensional bioprinting. The OBB matrices exhibit the desired self-healing, viscoplastic behavior required for sacrificial writing into functional tissue (SWIFT). As an exemplar, we created a perfusable cardiac tissue that fuses and beats synchronously over a 7-day period. Our SWIFT biomanufacturing method enables the rapid assembly of perfusable patient- and organ-specific tissues at therapeutic scales.
Project description:Recent years have witnessed the expansion of tissue failures and diseases. The uprising of regenerative medicine converges the sight onto stem cell-biomaterial based therapy. Tissue engineering and regenerative medicine proposes the strategy of constructing spatially, mechanically, chemically and biologically designed biomaterials for stem cells to grow and differentiate. Therefore, this paper summarized the basic properties of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. The properties of frequently used biomaterials were also described in terms of natural and synthetic origins. Particularly, the combination of stem cells and biomaterials for tissue repair applications was reviewed in terms of nervous, cardiovascular, pancreatic, hematopoietic and musculoskeletal system. Finally, stem-cell-related biomanufacturing was envisioned and the novel biofabrication technologies were discussed, enlightening a promising route for the future advancement of large-scale stem cell-biomaterial based therapeutic manufacturing.
Project description:Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.