Poster abstracts | Biomanufacturing Opportunities Workshop

Authors: Farah Deeba, David W. Wood and Davita Watkins (William G. Lowrie Department of Chemical and Biomolecular Engineering), and Chad Rappleye (Department of Microbiology)                                             

Abstract: Emerging resistance to chemical fungicides and negative environmental impacts have compelled researchers to evaluate alternative methods to control disease-causing fungi. Among various food safety methods, chitinolytic enzymes provide a safe and cost-competitive natural (biological) approach to controlling fungi as compared to chemical fungicides, but the main disadvantage of enzymes relies on their instability, which decreases their half-life in the case of temperature and pH fluctuations. Enzyme immobilization can offer an opportunity to enhance enzyme stability and reusability. In this scenario, waste-derived nanoparticles (NPs) are considered ideal for synthesizing sustainable, green, highly stable nano-biofungicides. Enzyme immobilization using biopolymer strategies is considered efficient in enhancing chemical and physical properties, high surface specificity, and stability of enzyme formulation. In this study, we are synthesizing low-cost, nontoxic, environmentally friendly chitinase immobilized (NPs) based on (1) Poly (ethylene glycol)-poly(caprolactone) (PEG-PCL) and (2) Agro-waste-derived polymers (Pullulan).  

See the poster

Authors: Seulhee Kim (Department of Biomedical Engineering), Chen Kai (William G. Lowrie Department of Chemical and Biomolecular Engineering), Lufang Zhou (Department of Biomedical Engineering, Department of Surgery), and Xiaoguang “Margaret” Liu (William G. Lowrie Department of Chemical and Biomolecular Engineering)

Abstract: In the past few decades, Adeno-Associated Virus (AAV) has been proved to be the safest and most effective virus vehicle in gene therapy. Furthermore, it was modified to recombinant AAV to suit better for clinical applications owing to its low toxicity to have stable long-lasting expression. Adherent HEK293 human cell line has been widely used for AAV production despite its limited ability to scale up. Meanwhile, large-scale AAV production from bioreactor, a type of suspension culture, has been explored recently. Considering the advantages of suspension cultures, it highlights the potential of cost effective AAV production process. This study aimed to compare three different suspension cultures, from shaker flask, spinner flask, to bioreactor, and evaluate the obtained AAV’s quality. The average titer obtained was approximately 5E+10 vg/ml, and the bioreactor showed its capability to yield the highest titer among the three methods. In addition, purification methods were developed using anion exchange based Next Generation Chromatography (Biorad) as the purity of AAV is a crucial deciding factor for the success of gene therapy. Furthermore, biochemical analysis and IVIS imaging were conducted for the virus quality and control. 

See the poster

Authors: David W. Wood (William G. Lowrie Department of Chemical and Biomolecular Engineering) and Izabela Gierach (Protein Capture Science)

Abstract: The goal of the next generation of biomanufacturing of the peptides, enzymes and other proteins is to eliminate complexity and streamline production of high-purity products in a scalable and highly controllable way. Therefore, a breakthrough purification platform known as iCapTag was created for a wide range of applications. This platform results in on average 95-99% purity of products after usage of a single purification column and significant reduction of purification steps. This platform can be used for manufacturing of enzymes, hormones, peptides, fragments of proteins, biosimilars, and novel biopharmaceuticals. Few examples are here presented, including expression and purification of proteins and protein fragments for industrial use. Currently, the iCapTagTM platform is used in academic, governmental and biotech labs and is tested for future large-scale GMP productions. Partnerships and collaborations across wide range of fields are welcome. 

See the poster

Authors: Peter E. Beshay and Melika Shahhosseini (Department of Mechanical and Aerospace Engineering), Jonathan W. Song (Department of Mechanical and Aerospace Engineering and Comprehensive Cancer Center), Carlos E. Castro (Department of Mechanical and Aerospace Engineering and Biophysics Graduate Program)

Abstract: It is well appreciated that reciprocal communication between cells and the surrounding extracellular matrix (ECM) can modulate the properties and the behavior of both the ECM and cells to influence both form and function. For example, modulation of the mechanical properties of the ECM by cells is believed to significantly influence the progression of certain diseased tissue such as fibrosis, healing wounds, or the stroma of tumors, all of which are known to exhibit ECM remodeling through the cross-linking of fibrillar collagen and/or deposition of non-collagenous ECM. DNA-based molecular sensors have been of particular interest for studying cell interactions due to their high tunability, sensitivity and easy integrability into various biological systems. Here, we present hybrid systems that utilize microfluidic devices, tissue models, and DNA-based nanoscale sensors that enable monitoring live cell and tissue interactions with molecules in their surrounding environment.

Authors: Tatiana Z. Cuellar-Gaviria (Department of Biomedical Engineering), María A. Rincon-Benavides (Department of Biomedical Engineering, Biophysics Program) Natalia Higuita-Castro (Department of Biomedical Engineering, Biophysics Program, Department of Neurosurgery)

Abstract: The use of engineered extracellular vesicles (EVs) as next-generation non-viral nanocarriers for gene and drug delivery applications is a promising strategy. EVs are cell-derived carriers that share molecular cues with the donor cell and play a crucial role in cell signaling under healthy and pathological conditions. The implementation of engineered EVs as novel carriers of therapeutic payloads help to circumvent major practical and translational barriers inherent to other delivery methodologies such as capsid size restrictions and redosing limitations for viral vectors, and low transfection efficiencies for synthetic carriers. Moreover, EVs possess an innate ability to penetrate biological barriers, have low immunogenicity, and higher stability in biofluids compared to synthetic carriers. Nonetheless, the translation of engineered EV-based therapies in the clinic has been hindered by a variety of manufacturing constrains including low yield, heterogeneity, scalability problems, and limitations in terms of high-throughput characterization methods. We describe the implementation of nanotechnology-enabled biomanufacturing approaches designed to streamline the production of engineered EVs for targeted gene delivery to the brain and to enhance wound healing. All this postulating engineered EVs as next generation non-viral nanocarriers for molecular cargo delivery and gene therapy applications. 

Authors: Ademola Duduyemi (presenting author, Department of Animal Sciences), Emmanuel Okezie and Francisca Udegbe (Department of Animal Sciences), and Thaddeus Ezeji (corresponding author, Department of Animal Sciences)

Abstract: To reduceworld’s over-reliance on petroleum-derived fuels and chemicals such as gasoline, butanol, and hydrogen (H₂), and decrease emission of greenhouse gases such as carbon dioxide (CO₂), it is important to produce these fuels and chemicals from renewable resources (e.g., biomass) using biocatalysts (microorganisms). Microbial production of these compounds at commercial scale, however, is hampered by several factors including small product titers and sub-optimal productivities stemming from the toxicity of compounds to the producing microorganisms. Additionally, greenhouse gas (CO₂) is often produced during microbial fermentation processes. Our research team has been developing different technologies for real time recovery of fuels and chemicals from the broth during fermentation so as to keep concentrations of these compounds in the bioreactor below the threshold of toxicity or inhibition to the biocatalyst actions, and allowing fermentation to proceed unimpeded until all the substrates present in the bioreactor are utilized and converted to target compounds. Additionally, our research team is developing processes for H₂ production from biomass, and conversion of greenhouse gas, CO₂, to fuels and chemicals such as butanol. Can these technologies be developed as standalone processes or integrated into existing biorefinery operations? These areas and more will be highlighted during the poster presentation at the Ohio State Biomanufacturing Opportunities workshop. 

Authors: Diego Alzate-Correa and Ana Isabel Salazar-Puerta (Department of Biomedical Engineering) and Daniel Gallego-Perez (Department of Biomedical Engineering and Department of Surgery)

Abstract: Tissue biomanufacturing has the potential to facilitate the development of highly promising personalized cell therapies for regenerative medicine. Current reprogramming methodologies, however, face major practical and translational hurdles, including heavy reliance on viral transfection, and a vastly stochastic nature, which often leads to inefficient and/or unpredictable reprogramming outcomes. We developed novel nanotechnology-based approaches that overcome these barriers by enabling deterministic transduction of reprogramming factors into cells and tissues without the need for viral vectors. In addition, cell and tissue nano-transfection (TNT) promotes remarkably fast and efficient direct cellular reprogramming with high adaptability for in vitro, in vivo, and ex vivo applications. Such platform technologies could be applicable to virtually any reprogramming model, and its minimally disruptive and non-viral nature make it an ideal candidate for use in complex diseases. Ongoing studies in our research program has focused on the implementation of nanotransfection-driven strategies to enable reprogramming-based cell therapies for conditions such as Alzheimer's disease, stroke, ischemic necrosis, peripheral nerve injury, metabolic disorders, diabetes, and cancer.

Authors: Young Ngo and David Nagib (Chemistry and Biochemistry)

Abstract: Our research is focused on harnessing the untapped reactivity of abundant chemical  motifs, such as C-H and C-O bonds - that are typically considered inert - to enable: (1) late-stage functionalization of medicinally relevant molecules, or (2) bio-orthogonal couplings of proteins and nucleotides. To this end, we have recently developed new approaches for selective C-H and C-O functionalization of alcohols, amines, and carbonyls, using a combination of open and closed shell processes that act in concert with one another. These radical- and carbene- mediated strategies have enabled discovery of new classes of reactivity to streamline the synthesis of complex molecules with biological and industrial significance. 

Authors: Thomas DePalma, Kennedy Hughes and Marie Tawfik (Department of Biomedical Engineering), Colin Hisey (Department of Chemical and Biological Engineering), and Aleksander Skardal (Department of Biomedical Engineering, Comprehensive Cancer Center)

Introduction and Objectives:

Astrocytes are involved in many of the essential processes in the CNS including maintenance of synapses, regulation of the blood brain barrier, and injury response. Astrocytes transition to a reactive state in response to trauma or tissue damage. This reactive state is observed in almost all brain diseases including brain cancers. It is evident that reactive astrocytes can be both destructive and aid in recovery of the CNS, and the classification and causes of these different reactive states is an ongoing subject of research. In glioblastoma, an aggressive form of primary brain cancer, it has been observed that tumor associated astrocytes can impact tumor cell proliferation and contribute the unique inflammatory environment of the tumor. Currently there are few systems that allow for the culture of primary human astrocytes in 3 dimensions which limits our ability to study GBM-Astrocyte interactions in a physiologically relevant environment. Astrocytes grown in 2D and in commonly used 3D extracellular matrix (ECM)-derived hydrogels such as Matrigel or collagen I display markers of activation making it difficult to study the drivers of reactivity and the downstream impacts of cell activation. In this study, we build off our previous work developing 3D hydrogel biomaterials to develop a system that permits the study astrocyte reactivity in vitro. The hydrogel system must maintain astrocytes in a quiescent state and allow for transition to a reactive state in conditions like those present in disease conditions.

Methods: All hydrogels tested are composed of natural materials that are chemically modified to allow for UV initiated chemical crosslinking. Collagen-HA hydrogels are composed of methacrylated collagen and thiolated hyaluronic acid (HA). HyStem gels are composed of thiolated HA, thiolated gelatin, and polyethylene glycol diacrylate (PEGDA). The brain-specific ECM hydrogel(bsECM) is composed of methacrylated HA (HAMA), methacrylated Gelatin, and PEG dithiol (PEGdt). Storage modulus, loss modulus, and stress relaxation characteristics are measured using rheology. Primary human astrocytes (ScienCell) are seeded within the hydrogels at a density of 1-2 million cells/mL. 10uL droplets of hydrogel are crosslinked with 2 seconds of UV light. Cells are grown for 4-10 days in astrocyte medium (ScienCell) before analysis. Astrocyte reactivity is analyzed using the following methods. Hydrogel constructs are fixed using 4% PFA before immunostaining. Images are acquired using confocal microscopy. Image analysis is conducted to quantify cell morphology, protein expression and location using custom programs in MATLAB and Nikon Elements Software. To isolate RNA, hydrogel samples are first dissolved in collagenase-hyaluronidase mixture for 20 minutes before isolation with TRIzol reagent. rtPCR is conducted to analyze expression of genes associated with astrocyte reactivity and inflammation. Astrocytes grown in normal astrocyte medium (sciencell) is compared with growth factor cocktail known to induce A1 reactive astrocytes (media supplemented with Il-1α , TNF and C1q), and conditioned media from several GBM cell lines (U87, A172, U373, U87 EGFRIII, BT169).

Results: Mechanical analysis of all hydrogels used in these studies, Col-HA, HyStem, and bsECM hydrogel reveals that they all have a storage modulus that matches that of the human brain, 50-500 Pa. Confocal imaging reveals that astrocyte spreading is increased in the bsECM hydrogel and Col-HA compared to HyStem. Image analysis reveals lower GFAP and vimentin expression in the brain hydrogel indicating that the astrocytes are less reactive in this environment compared to other conditions tested. rtPCR is currently underway to further confirm these results. Activation of astrocytes using the growth factor cocktail of Il-1α , TNF and C1q is also used to induce the reactive astrocyte phenotype in the brain hydrogel. Studies using conditioned media from several GBM cell lines is currently underway. Results from these studies should reveal whether different cancer cell populations have different impact on the reactive state of astrocytes in the nearby tumor microenvironment.

Conclusion: Our studies have shown that this bioengineered brain-specific ECM hydrogel composed of HAMA, GelMA, and a PEGdt crosslinker promotes the morphology and function of astrocytes observed in vivo. We have also begun to show that astrocytes grown within the hydrogel can be activated and used to study how astrocytes change in different disease states, specifically glioblastoma. Future studies will include co-cultures of tumor cells and astrocytes in the constructs to determine whether physical interactions of the cells impact astrocyte phenotype and/or cancer cell growth. Other cell types such as microglia, pericytes, and endothelial cells will also be added to further investigate the ways in which GBM associated inflammation affects cell function and behavior at the blood brain barrier.

Authors: Ivan S. Pires, Donald A Belcher and Andre F. Palmer (William G. Lowrie Department of Chemical and Biomolecular Engineering)

Abstract: Current methods for protein purification require extensive process development as well as cumbersome and costly unit operations. Herein, we describe a simple protein purification strategy. The approach exploits molecular size changes in order to isolate a target protein (TP) from a complex mixture via tangential flow filtration (TFF). Essentially, a TP binding molecule (TPBM) is used to selectively alter the size of the TP and that change in size between the TP and TP-TPBM complex enables purification of the TP-TPBM complex from the complex mixture. Upon purification of the TP-TPBM complex, the TP can be dissociated and isolated. This process was demonstrated to recover human serum albumin (HSA) in its complexed form from an artificially produced mixture of hemoglobin (Hb) and HSA and from plasma using an anti-HSA polyclonal immunoglobulin G (IgG) as the TPBM. Moreover, haptoglobin (Hp) in its complexed form was recovered from human Cohn Fraction IV using Hb as the TPBM. The Hb-Hp complex was then partially dissociated with 5M urea to yield Hp. Furthermore, we developed and demonstrated that a simple mathematical model may be used to describe the TFF purification process and guide process development. Taken together, this technology and mathematical tool present a new approach for selective purification of proteins using TFF and simple mathematical model to describe and predict the performance of TFF systems for protein separations. 

See the poster

Author: Aleksander Skardal (Biomedical Engineering)

Purpose/Objectives: Regulator of calcineurin 1, isoform 4 (RCAN1-4) has been shown suppress metastasis in thyroid cancer. RCAN1-4 knockdown increases proliferation in vitro and metastatic burden in vivo, evidence that the gene regulates later steps in the metastatic cascade and potentially impacts metastatic dormancy. To study RCAN1-4 in thyroid-to-lung metastasis, a metastasis-on-a-chip (MOC) model was developed using control and RCAN1-4 knockdown clones of thyroid cancer cell lines hTh74 and FTC236.
Methodology: Fluorescently tagged cancer cells were circulated through microfluidic channels, running parallel to lung hydrogels and allowing tumor cell-lung tissue interactions. Imaging and quantification measured tumor cell invasion distance and number for control and knockdown clones of each cell line. Proteomics of hTh74 and FTC236 lines identified changes to invasion and dormancy-linked pathways.
Results: RCAN1-4 knockdown increased invasion distance into lung hydrogels, as well as number of invaded cells. Differing endogenous RCAN1-4 expression between the cell lines was reflected in a greater difference between knockdown and control hTh74 invasion as compared to FTC236, aligning with in vivo observations. RCAN1-4 knockdown did not change the time between tumor cell introduction to the system and attachment to lung sites, implying that RCAN1-4’s metastasis suppressing functions may be more related to motility and proliferation than extravasation.
Conclusion/Significance: Here, we demonstrated alignment of metastasis-on-a-chip results with in vivo observations while exploring more in-depth analyses of RCAN1-4's metastasis-suppressing roles. This model aims to overcome difficulties in monitoring distinct metastatic steps and kinetics in animal models, as well as provide a fully human alternative for improved translation.

Authors: Ivan S. Pires, Donald A Belcher and Andre F. Palmer (William G. Lowrie Department of Chemical and Biomolecular Engineering)

Abstract: While there are many existing techniques for purification of apoproteins, these approaches have flaws such as the use of harsh denaturants, highly flammable solvents, low yields, cumbersome manufacturing processes, and high capital and operating expenses. Furthermore, the techniques are not easily scalable, which limits widespread application. Herein, we describe a general method for separating small hydrophobic ligands from proteins facilitated by tangential flow filtration (TFF), such as heme from hemoglobin (Hb) or metabolites and lipids from human serum albumin (HSA). More importantly, this novel approach does not require the use of specialized equipment, is relatively inexpensive, and easily scalable. Furthermore, this technology has the potential to be applied to any ligand-associated protein to produce the resultant apoprotein. We demonstrate the feasibility of this technology by extracting the high affinity (KD~10 pM) heme ligand from Hb to yield apohemoglobin (apoHb). The process underwent scale-up without major alterations in overall protein yield or protein bioactivity. Overall, large-scale manufacturing of apoproteins opens-up a new approach to facilitate small molecule drug transport in vivo. Furthermore, these proteins may be used for targeted drug delivery, bioimaging, and/or scavenging of toxic molecules during various disease states. 

See the poster