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Downstream Processing: Non-Chromatography Based Separation of Biomolecules: Non-Chromatography Based Separation of Biomolecules
02:00pm - 03:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Mahsa Rohani, Presider, ‍ ; Akshat Gupta, Presider
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Protein purification methods based on mechanical separations like centrifugation, hydro cyclones and elutriation along with depth filtration, multi-phase partitioning, precipitation, flocculation, and crystallization are widely used in biopharmaceutical industry. These techniques enable, enhance and complement many key and novel separations required for purification of biomolecules and are being actively studied and improved in order to meet evolving needs of industry and a higher demand for performance. This includes effective harvesting of higher density cell cultures; enhanced impurity clearance; enhanced performance of chromatography, sterile and virus filtration steps; in stand-alone, integrated, or continuous/semi-continuous manner. These technologies also play a pivotal role in identifying novel ways of using conventional unit operations to solve both current and future bioprocessing challenges of complex biological products. This session seeks to report advances in the development, fundamental understanding, and industrial application of non-chromatographic, non-membrane-based unit operations to achieve desired bio separations, as well as cases demonstrating the advantages/disadvantages of integrated processes thereof. Operations of interest may include; traditional unit operations, centrifugation, flocculation, depth filtration or less traditional unit operations, hydro cyclones, elutriation, acoustic separation, aqueous multi-phase partitioning, precipitation, crystallization and polymer-aided flocculation. In addition, we would like to welcome both experimental and modeling submissions. Priority will be given to those that provide insights and present approaches of general utility, and for whom experimental and/or manufacturing implementations are presented and compared with alternative approaches.

Monday
Depth filter media and post viral inactivation filtration performance
02:00pm - 02:20pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Downstream mAb processing typically employs a harvest step, multiple chromatography steps, multiple process intermediate filtrations and a final ultrafiltration step. Each unit operation in the process is designed for specific purification goals based on the quality of the incoming feed stream and the desired quality profile of the drug substance. Advances in upstream processing are generating more productive cell cultures processes. This is often accompanied with increases in product impurities, such as aggregates, and process related impurities, such as residual host cell proteins (rHCP). These purification challenges can only be overcome if each unit operation is providing the most robust impurity removal possible.
The work presented here highlights the efforts to understand how the choice of post viral inactivation filter media impacted overall process impurity clearance which can ultimately lead to a more stable drug substance. The filter evaluation included 6 filters, from 4 different manufacturers, with 5 unique media types, conducted with 4 different mAbs. Several performance (loading and yield) and quality attributes (turbidity reduction, aggregate & residual impurity clearance) were compared across the panel of filters tested. Additionally, each filter was evaluated for its ability to clear impurities related to PS80 degradation. We observed that tracking rHCP impurity clearance across filters provided a primary means of differentiation and ultimately guided final filter selection.

Monday
Single-use fluidized bed centrifugation as an intensified platform approach for clarification
02:20pm - 02:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Mr. Martin Saballus, Presenter, Sartorius; Markus Kampmann
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The growing demand for biopharmaceuticals has resulted in the development of intensified processes using highly productive cell lines and high cell density cultures. Accompanied by large amounts of biomass and dissolved host cell impurities, such processes increase the requirements for clarification of the cell culture fluid (CCF). Due to the lack of capable process platforms, clarification has been identified as a bottleneck.

Therefore, a novel approach for clarification applying a single-use fluidized bed centrifuge (FBC) followed by a small filtration step was investigated. The approach was evaluated for a broad range of mammalian cell concentrations (20 – 110 million cells/mL) for production of a monoclonal antibody (mAb), providing insights on the challenges associated with harvesting intensified CCF.

High mAb recoveries of 90 – 100 % and low turbidities in the harvest pool were obtained, showing the suitability of the approach for various CCF. Moreover, a model was developed to calculate CCF throughput of the FBC as a function of feed cell concentration.

To achieve additional removal of dissolved impurities, which represent a high burden especially in low viable cell cultures, pretreatments of CCF prior to FBC clarification by low-pH precipitation or cationic polymer flocculation were investigated. For both approaches, particle formation, processability of the pretreated CCF, impurity reduction and quality of mAb were examined. It was demonstrated that both pretreatments facilitate the removal of cell debris and dissolved DNA due to their aggregation. This resulted in up to 4x higher filter capacities in the post-FBC filtration step and > 90 % DNA removal in the clarified harvest pools.

Therefore, the presented FBC based approach has a great potential to be used as a clarification platform for various CCF to intensify the removal of cells and impurities, thus streamlining the subsequent purification process.

Monday
Mechanistic modelling of Chinese hamster ovary cell clarification using acoustic wave separator
02:40pm - 03:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Mr. Shantanu Banerjee, Presenter, Indian Institute of Technology Delhi; Mohammad Areeb Afzal; Paresh Chokshi; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Acoustic wave separation (AWS) is gradually emerging as an effective method for continuous clarification of Chinese Hamster Ovary (CHO) cells. Here we present a mechanistic model for AWS based on population balance model (PBM) along with acoustic agglomeration kernel to predict cell size distribution as a function of time. Cell trajectory in the acoustic zone has been analysed numerically based on inertial and acoustic forces. The results indicate a significant contribution of flowrate and acoustic power on cell dynamics in the acoustic zone. Using this understanding, PBM has been used to determine the agglomeration rate and cell separation efficiency (CSE) based on the flowrate, acoustic power, load cell density and cell diameter. Results show that more than 90% CSE was achieved for flowrates < 5 mL min-1 and acoustic power > 12.5 W. Higher load cell densities also resulted in enhanced separation owing to the increase in inter-cell collisions. Multiple chambers increased the residence time of cells and led to an increase in the overall CSE. Experimental results confirm that the mechanistic model offers an accurate prediction and a better understanding of the CHO cell agglomeration process using the novel AWS technology. Furthermore, a generalized correlation has been developed using the Buckingham Pi theorem between the dimensionless numbers composed of relevant process and geometrical variables to predict the agglomeration efficiency. To our knowledge, this is the first such study to model the agglomeration dynamics of CHO cells in acoustic standing waves.
Monday
Index of floc strength (FSI) as determinant of robust bioprocess options
03:00pm - 03:20pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Sofia Nunes, Presenter, UCL; Andrea Rayat, University College London
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In the recovery of biological products, flocculation is often used to aid primary recovery steps such as cell harvest and clarification of homogenates. It is important to generate large strong flocs to avoid floc breakage that could reduce the performance of subsequent bioprocessing steps. Therefore, flocculation performance must be characterised based on both floc growth and strength resulting in robust flocs.
This work presents an evaluation of floc growth and strength based on the analysis of particle size distribution (PSD) changes during bioprocessing. This novel technique aims to address the limitations of the typical measurement of floc strength by mixing studies and to overcome the challenges associated with the manual observation of PSD changes.Using the kompAs™ shear device in tandem with PSD measurements, the PSD data of the flocs studied at different conditions are converted to unidimensionalindex of floc strength (FSI). PSD is measured at both ends of the flocculation process and after shear experiments wherein the flocs are exposed to process shear, similar to the levels encountered during industrial operations, such as centrifugation. A sequential data analysis is performed on the PSD from which FSIsare derived.
The application of this novel approach was demonstrated in scale-down investigations to quantify and evaluate floc growth and strength using different flocculation experimental conditions for E.coli homogenates to produce an industrially-relevant enzyme. It was also used to assess scale-up strategies by comparing ultra scale-down and pilot scale flocculation. The FSI analysis provided insights on the process and agreed with the results obtained from turbidity data, clarification performance and floc characteristics. The established approach of combining ultra scale-down experiments using kompAs™ and the novel FSI analysis enables the modelling of high throughput flocculation studies to inform bioprocess options for primary recovery development.
<b>Novel studies to prepare and analyse PSD</b> to evaluate floc growth and strength.

Novel studies to prepare and analyse PSD to evaluate floc growth and strength.


Monday
Design of a tailor-made aqueous two-phase system (ATPS) and its intensification by adding excipients
03:20pm - 03:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The performance of biocatalysts in evolving green processes increasingly reaches industrial competitiveness. During enzyme manufacture, after protein expression, aqueous two-phase extraction (ATPE) using an aqueous two-phase system (ATPS) is a promising and sustainable alternative to common, cost-intensive chromatographic purification. Instead of the common state-of-the-art ATPS design based on “trial-and-error” screening approaches, we here demonstrate our method for ATPS design to an industrially relevant amine dehydrogenase (AmDH). The method includes thermodynamic modeling of ATPS phase composition and investigating conformational and colloidal protein stability to rapidly select a tailor-made ATPS, offering optimal process and processing conditions. All investigations and the selection of the sodium glutamate (MSG)-polyethylene glycol (PEG) 2000 ATPS were performed in less than four weeks, demonstrating proof of concept for a fast and sustainable ATPS selection in an early stage of downstream processing development.
ATPS phase formers often reduce conformational and colloidal stability of the target protein, leading to aggregation or precipitation, and thus product loss. We demonstrate that this remaining drawback can be overcome by selecting suitable excipients to increase conformational and colloidal stability, improve solubility of the target biomolecule, and lead to an overall increase in extraction performance. We confirm transferability of this approach with AmDH and Immunoglobulin G (IgG) purification. In a model ATPE system (no impurities) with L-arginine as excipient, precipitation of AmDH can be decreased to increase yield from 78.7 to 87.9 wt%. From crude extract, 79.1% yield in the desired top phase was achieved, with < 7% impurities. We expect that addition of excipients to ATPS will broaden the process window and simplify handling during downstream processing.

New Technologies for the Delivery & Targeting of Therapeutics: New Technologies for the Delivery & Targeting of Therapeutics
02:00pm - 04:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Yonghyun Kim, Organizer, The University of Alabama; Maryam Raeeszadeh Sarmazdeh, Organizer, University of Nevada, Reno; James Van Deventer, Presider, Tufts University; Kevin Dooley, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

New technologies for delivering therapeutic constructs to specific cell types and tissues are necessary to unlock the potential of precision medicines and expand the therapeutic index of existing interventions. This session will focus on targeted delivery strategies currently being developed in both academia and industry for modalities including viral vectors, nanoparticles, proteins, nucleic acids, and small molecules. Other relevant topics for this session include enhancing endo-lysosomal escape/intracellular delivery, extending circulating half-life, design of novel delivery vehicles, discovery and optimization of targeting ligands, and strategies for controlling cargo release kinetics.

Monday
Peptide-mediated growth factor gene therapy using extracellular matrix-inspired biomaterials
02:00pm - 02:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Chronic wounds are a significant worldwide problem with enormous health-related and economic impacts. These adversities are particularly striking in patients with diabetes mellitus, who have an estimated lifetime incidence rate of 19-34%, with a cost of total patient care of ~$25B. Two key issues hindering repair include limitations in growth factor activity and accessibility, and the prevalence of infection and biofilm formation in the chronic wound environment. Our studies demonstrate a new approach with a high likelihood to overcome these issues through application of collagen mimetic peptides (CMPs) as drivers of growth factor gene therapy and drug delivery during wound healing. Local gene therapies offer compelling potential advantages for improved growth factor delivery due to their modularity and inherent benefits in protein stability, protein bioactivity, sustained release, and cost, as well as the relative ease of delivering multiple genes and the spatial control achievable through use of tissue-specific promoters. CMP gene therapies, in particular, provide exceptionally stable and tunable links between DNA nanocarriers (polyplexes) and collagen, leading to localized and efficient expression of growth factors including PDGF and VEGF, even after prolonged exposure to serum. This talk will highlight the design principles underlying CMP gene scaffold design for application in wound repair, along with results demonstrating the efficacy of CMP-PDGF and CMP-VEGF gene therapies for inducing faster wound closure with minimal scar formation as well as improved collagen production, myofibroblast activity, collagen orientation, and angiogenesis in murine wounds. We also highlight new work showing the benefits of CMPs for controlling the delivery of multiple gene and/or drug therapeutics in the wound environment to enable holistic wound management. These results are indicative of the potential of matrix-modified biomaterial scaffolds in effective treatment of chronic non-healing wounds.
Monday
Design and characterization of a platform for efficient intracellular protein delivery
02:40pm - 03:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Dr. Wei Lv, Presenter, Georgia Institute of Technology; Anshul Dhankher; Talmage Studstill; Dr. Julie Champion, Georgia Institute of Technology
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The clinical translation of many therapeutic proteins is limited by their poor cellular permeability. Enabling intracellular delivery of bioactive proteins could expand therapeutic options for many currently undruggable targets. Although progress has been made in developing protein intracellular delivery carriers, mainly by utilizing nanoparticle-based system, they face challenges of low loading, cytotoxicity and endo-lysosomal entrapment. To address these challenges, we previously developed a protein self-assembly drug carrier (Hex), comprised of a hexameric coiled coil and antibody binding domain, that can deliver antibodies to intracellular targets. Here, we systematically studied the trafficking of the Hex-antibody complex in living cells and used different variants of the Hex carrier to understand the function of each component in intracellular protein delivery. We demonstrated Hex-antibodies were internalized through multiple endocytic routes and provide evidence of endo/lysosomal disruption and Hex-antibody release to the cytosol. We also discovered that greater exposure of the coiled coil hexamer promoted internalization into cells and that the presence of histidine tags promoted endosomal disruption. Further, we determined that Hex delivery can be extended to proteins beyond antibodies. The therapeutic potential of the platform is demonstrated by manipulating a disease-relevant therapeutic transcription factor, STAT3, to inhibit its nuclear translocation. This study has generated understanding of key steps and components in the efficient cytosolic therapeutic protein delivery and will facilitate the development of applications in both the therapeutic nanoassemblies and research tools.

Monday
Withdrawn
03:00pm - 03:20pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Monday
Withdrawn
03:20pm - 03:40pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Monday
Rational design of NIR controlled brain targeting drug delivery vehicle for neuro-regeneration purpose
03:40pm - 04:00pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Treatment of brain diseases remains a challenge due to difficulties of therapeutic agents' delivery to the brain. Specifically, the application of hydrophobic neuro-regenerative drugs was limited due to their poor bioavailability caused by insolubility and short half-life, and lack of effective targeting and controllable release. Herein, we presented our study on developing a novel 808nm near-infrared (NIR) responsive drug delivery vehicle, the core-shell superparamagnetic iron oxide-hollow gold nanoshell (SPIO@HGNS) functionalized with positively charged porous coordination cages (PCCs). The 808nm NIR light matches HGNS's surface plasmon resonance and has the excellent tissue penetration ability to reach the brain area. The ion-exchange resin (IER) mediated surface modification by (11-Mercaptoundecyl)-N, N, N-trimethylammonium bromide (MUTAB) tuned the overall surface charge from negative to positive, which enabled the encapsulation of anionic retinoic acid (RA). The dose-dependent improvement of RA loading was revealed by zeta potential and NMR results. Higher drug loading efficiency was achieved comparing to pure HGNS or SPIO/Au nanoparticles. Uniform nanoparticle dispersity was also observed by TEM. The in vitro release of RA was performed under the stimulation of 808nm (100mW/cm2) NIR ultrafast LASER at an interval of 3 h with each time NIR-exposure of 10 min. The RA release kinetics was evaluated by measuring the absorbance of RA via UV-Vis. Results revealed that RA's higher release rate was triggered by the stimulation of NIR light, demonstrating the light-controllable drug release kinetics. The proposed SPIO@HGNS nanocarrier has the potential for the controlled drug release in the targeted brain site because of the magnetic nature itself and the NIR-responsive drug release mechanism.
Engineering Microbial Communities & Non-Model Systems : Engineering Microbial Communities & Non-Model Systems
04:30pm - 06:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Mark Mimee, Presider, ‍ ; Michael Köpke, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Microbial communities and non-model organisms are increasingly used as production platforms for a wide range of biofuels, biochemical and biopharmaceuticals or in biomedical and bioremediation applications. The unique metabolism and physiology of non-model hosts can complement traditional hosts such as E. coli and yeast and address limitations including catalytic capabilities and overall productivity, while microbial communities are being exploited to enable robust performance under demanding industrial conditions. Several non-model organisms (e.g. Bacillus, Clostridium, Corynebacterium, Lactobacillus, Zymomonas) are already employed industrially for their ability to grow autotrophically, novel metabolic reactions, pathways and products, or high tolerance for common inhibitory compounds. These new hosts and communities are being engineered to access important feedstocks like lignocellulosic biomass, syngas, methane, methanol, glycerol, electricity, and carbon dioxide (amongst others), to increase sustainability, to enable novel biomedical and bioremediation application, and to decrease production costs. This session will focus on the recent developments in engineering non-model hosts and microbial communities for the production of biopharmaceuticals, biofuels, bulk chemicals and value-added specialty chemicals or in biomedical and bioremediation applications. Relevant topics include molecular and genetic parts and tool development, application of high-throughput approaches, pathway and community engineering, engineering community composition and optimizing divisions of labor, process development, and efforts to accelerate design-build-test loops through systematizing workflows, machine learning and other approaches. We welcome both industrial and academic contributors.

Monday
Utilizing CRISPR-Cas toolkits for genotype-phenotype discovery and engineering of non-model microbes
04:30pm - 05:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Carrie Eckert, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Our ability to efficiently engineer microorganisms to produce desired chemicals at industrially relevant scales, rates, and titers requires a combination of adaptive evolution and rational design methods to screen or select for mutants with optimized phenotypes. While useful, these methods are generally limited by the ability to introduce synthetically designed, non-biased, trackable mutations to clearly identify gene-to-trait relationships. With the advent of CRISPR genetic tools in our favorite model systems such as E. coli or S. cerevisiae, we have made progress towards the development of methods that enabled highly multiplexed and targeted mutagenesis libraries to allow for the rapid identification of genotype-to-phenotype relationships under selective conditions such as growth, production of desired compounds, and tolerance. Unfortunately, the adaptation of these methods for use in non-model systems with desired complex phenotypes that are not easily tractable (e.g. photosynthesis, biomass degradation, CO/CO2 fixation, low pH growth, etc.) is still lacking due to limited basic genetic tools and low transformation/editing efficiencies. In addition, when utilizing CRISPR tools, there can be widely varied success due to a lack of understanding of all of the caveats that affect how different systems will function in each host. I will discuss current progress for the development of expanded CRISPR-based genome engineering tools in some of the non-model microbes we work with in our group to enable accelerated gene-to-trait identification towards improving our understanding of these microbial systems.
Monday
CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria
05:10pm - 05:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Yasuo Yoshikuni, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products’ roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.
Monday
Uncovering the roles of stereochemistry in microbial chemical communication
05:30pm - 05:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Prof. Akira Kawamura, Presenter, Hunter College of CUNY; Alison Clare Domzalski, M.S., CUNY The Graduate Center/ CUNY Hunter College; Valeria Vigo; Jessica Malcolm; Liliana Margent
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Microbes use chemical signals for intraspecies, interspecies, and interkingdom communication. Studies on such chemical signals set the stage to learn the chemical language of microbes, which, if we can learn, would enable us to harness the immense metabolic power of microbes to address major issues in medicine, energy, and the environment. While several classes of chemical signals, such as quorum sensing factors, have been characterized, they probably represent a small fraction of the entire chemical lexicon of microbes. This is because the vast majority of microbes in nature are unculturable as an axenic culture (i.e., a culture of a pure microbial strain), which has been used to discover most of the known chemical signals. To gain access to previously overlooked chemical signals, we have been studying mixed microbial cultures (MMCs) that are derived from plants and environmental samples. Because MMCs are derived from communities of environmental bacteria, they reflect natural contexts wherein chemical crosstalks among microbes have direct ecological significance. Our recent study indeed uncovered a series of potential chemical signals, including 12-hydroxystearic acid, which has not been studied in the context of microbial communication. Furthermore, MMCs produce stereoisomeric mixtures of chemical signals, including (R)- and (S)-isomers of 12-hydroxystearic acid, suggesting the importance of stereochemistry in microbial communication. Here, we present our stereochemistry-activity relationship study on potential chemical signals discovered from MMCs, which aims at gaining new insights into the roles of stereochemistry in microbial communication. Implications of our findings with regard to biomedical, biotransformation, and bioremediation applications will be discussed.

Monday
Building genetic engineering tools for anaerobic gut fungi
05:50pm - 06:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Anaerobic fungi (Neocallimastigomycota) are native to the digestive tracts of ruminant and hindgut fermenting animals where they are central to the digestion of crude lignocellulosic materials. Anaerobic fungi secrete the largest known diversity of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom (>300 CAZymes), which unaided can degrade up to 60% of the ingested plant material. Thus these microorganisms have rich biotechnological potential ranging from developing renewable energy technologies to improving animal nutrition. To this end, we are working to build a suite of genetic engineering tools for these organisms. This tool box includes a variety of promoters, antibiotic resistance markers, fluorescent reporters, as well as methods to transform anaerobic fungi. We have also identified subcellular localization signal sequences for targeting proteins to specific organelles (e.g nuclear localization sequences). Currently we are working to isolate an Autonomously Replicating Sequence to enhance plasmid stability. Preliminary efforts show addition of these putative ARS sequences to unstable fungal plasmids rescues growth to nearly positive control levels. Simultaneously we are working to identify a minimal centromeric sequence to develop a truly stable plasmid. By leveraging chromatin conformation capture (HiC) we have determined the degree of both structural and sequence conservation in gut fungal centromeres across 2 genera. Future efforts will involve testing these putative minimal centromeres in anaerobic fungi for assessing plasmid maintenance. Once validated, these parts will form the foundational tools to generate a deeper systems-level understanding of anaerobic fungal physiology while establishing fundamental knowledge about regulation of gut fungal CAZymes.
Monday
Elucidating metabolic and regulatory networks of non-model organisms for lignin valorization
06:10pm - 06:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 04
Jinjin Diao; Rhiannon Carr; Yifeng Hu; Prof. Tae Seok Moon, Presenter, Washington University in St. Louis
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Lignin represents a renewable resource whose bioconversion could displace petroleum-based processes (1). Research has been conducted to develop a hybrid platform to generate value-added bioproducts from lignin breakdown products (LBPs) obtained by thermo-catalytic depolymerization of lignin (2). Traditional model organisms are not well suited for converting LBPs that consist of various toxic aromatic compounds, but non-model organisms have been identified as ideal candidates. One such host is Rhodococcus opacus, which has demonstrated high tolerance to LBPs and the ability to improve its tolerance and consumption through adaptive evolution. However, the key challenges in such lignin upgrading include our limited understanding of microbial utilization of toxic LBPs at gene levels and limited tools to engineer this organism. To understand and maximize its metabolic potential, we have employed multi-omics approaches, providing a systems-level understanding of the complex metabolism of the wild-type and evolved strains (3-5). Additionally, we have developed a genetic toolbox for R. opacus engineering (6-8). Despite recent advances in our understanding of its versatile metabolism and available genetic tools, studies of its gene functions at gene levels are still lagging. We will discuss our recent efforts to facilitate functional studies of this non-model organism using our genetic toolbox and to engineer it for lignin valorization with muconate production as a demonstration example (9).
Downstream Processing: Membrane-Based Downstream Bioprocessing: Membrane-Based Downstream Bioprocessing
04:30pm - 06:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Mahsa Hadidi, Presider, ‍ ; Ranil Wickramasinghe, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Membrane-based separation techniques are essential for processing of a wide range of biopharmaceutical products including small molecules, monoclonal antibodies, vaccines, viral vectors, etc. These techniques are utilized in a wide range of applications in bioprocessing from cell harvest/clarification to virus removal, and product purification, concentration, and buffer exchange. Membrane-based techniques enable and complement many key and novel separations required for purification of biomolecules and are being actively studied and improved in order to meet a higher demand for performance, such as effective harvesting of higher density cell cultures, high-throughput virus filters, high-concentration formulation development, and/or integration of unit operations for continuous/semi-continuous manufacturing. These technologies also play a key role in identifying novel ways of using conventional unit operations to solve both current and future bioprocessing challenges of complex biological products such as use of new/modified membrane material and novel mode of operations. This session seeks to focus on process understanding surrounding membrane operations and to report advances in the development, fundamental understanding, industrial application, and novel implementations of membrane-based unit operations to achieve desired bioseparations. Operations of interest include traditional and novel filtration and membrane processes for clarification, bioburden reduction, virus removal, ultrafiltration and diafiltration, formulation, etc. Both experimental and modeling (mechanistic, statistical, hybrid, etc.) submissions are welcome to this session. Priority will be given to those presentations that provide insights and present approaches of general utility, and for which experimental and/or manufacturing implementations are presented and compared with alternative approaches.

Monday
Evaluation of sterilizing grade membranes for liposome products using 3D imaging techniques
04:30pm - 04:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Two 3D imaging techniques, confocal microscopy and X-ray Computed Tomography (CT), were selected to understand the performance of liposome sterile filtration, relevant to medicines including vaccines and gene therapies. A dual layer polyethersulfone membrane was imaged following bacterial challenges, consisting of an asymmetric upstream membrane with a nominal retention rating (NRR) of 0.65 µm and a symmetric downstream membrane (0.2 µm NRR). Confocal microscopy was applied for identifying where liposomes (0.14 µm average diameter) and B. dim bacteria were retained within each membrane.
X-ray CT imaged the internal 3D structure of each membrane, making pore size measurements in agreement with vendor stated NRRs. The technique also enabled quantification of the asymmetry in the upstream membrane. Differential pressures between 0.7 and 2.1 bar were compared through positional retention analysis of liposome and bacterial fluorescence by imaging each membrane using confocal microscopy. Increasing differential pressure resulted in a sharper liposome retention peak closer to the surface of the upstream membrane. Using complementary 3D imaging techniques in this research enabled structural data from X-ray CT to be related with fluorescent retention profiles from confocal microscopy to identify the location of liposome fouling within the complex geometry of a sterilizing grade filter.

Monday
Economic evaluation of flow-through AEX column replacement by novel single-use polishing solution
04:50pm - 05:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Joris Van de Velde, Presenter, 3M
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Biopharmaceutical manufacturing processes for therapeutic proteins have shown rapidly increasing implementation of single-use technologies in recent years. These technologies can offer reduced capital investment and labor cost, higher flexibility and faster development and manufacturing cycles. The adoption of single-use polishing chromatography products for the replacement of resin-based columns has however proved to be challenging. Despite their clear advantages, these technologies have struggled with capacity constraints, high consumable costs and performance limitations for impurity and viral clearance.

We present an economic evaluation performed with Biosolve Process software, comparing the use of a resin-based column versus a novel single-use AEX technology, 3M Polisher ST. The modelling results demonstrate that this solution can effectively compete with column-based polishing steps and enable significant cost savings. The model teaches us which performance characteristics are required to make this possible, which subset of costs are most impacted and which process parameters strongly influence the overall result. Understanding these factors will avoid common misconceptions about single-use technologies and enable a successful transition from sequences of multi-use unit operations to intensified single-use process trains.

Monday
Evaluation of single-use tangential flow filtration technology in a conjugate vaccine manufacturing process
05:10pm - 05:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Highly effective vaccines against diseases caused by N. Meningitidis, S. pneumoniae, and H. influenza can be produced by chemical conjugation of the capsular polysaccharide from these bacteria to an immunogenic protein such as tetanus toxoid, CRM197 and diphtheria toxoid. The conjugation process typically involves a polysaccharide activation step in preparation for covalent linkage to the immunogenic protein. Tangential Flow Filtration (TFF) is widely used for buffer exchange and purification of activated and conjugated polysaccharides against reaction byproducts/residuals.
Over the past decade, single-use TFF technologies have emerged to reduce system preparation time, promote fast and flexible product change over and ultimately shorten process development and manufacturing time/cost. The primary objective of this work was to evaluate Pellicon® single-use TFF capsule against traditional reusable Pellicon® cassettes with respect to performance, flush requirement, product recovery and ease of use, whereas the secondary objective was assessing the scalability of Pellicon® TFF cassettes and capsule for purification of activated polysaccharides. In particular, the performance of a recently developed Pellicon® single-use TFF capsule was compared against traditional Pellicon® cassette by assessing TFF process performance (such as flux, residuals clearance, and yield) and post purification product attributes (such as concentration and mass-weighted average molecular weight). Good scaling was shown by comparing process performance and product attributes across different scales and formats (88 cm2 vs 1100 cm2 cassettes and 1000 cm2 capsule). Similar TFF process performance and post purification product attributes were observed for the single-use capsule and reusable TFF cassettes. The capsule requires a smaller flush than the cassette and is easier to use since it does not require a compression holder or pre-sanitization. The obtained results provide insight into the application of single-use technologies and scalability of TFF processes for purification of activated polysaccharides used in conjugate vaccine manufacturing.

Monday
Single-use redundant filtration (SURF) assembly and novel method of integrity testing
05:30pm - 05:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Santosh Rahane, Presenter, MilliporeSigma; Charles Raye; An-son Leong
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Pre-sterilized single-use assemblies containing redundant filter are commonly used for final filtration and filling operations where maintaining sterility is critical to assuring drug safety for patients. A novel single-use redundant filtration (SURF) assembly with Millipak® Final Fill (MPFF) filters as primary and redundant filters is designed to minimize the product losses at the final filtration stage. Multipurpose valve port of the MPFF filter enables aseptic connections for venting, sampling and connecting air-line for the purpose of integrity testing. As a result, the assembly contain less parts and connections, and has significantly lower hold-up volume compared to the traditional assemblies. Additionally, a method was devised to perform the integrity test of both primary and redundant filters using the valve port eliminating the need for filter(s) or bag(s) downstream of the redundant product filter traditionally installed for collecting test liquids and venting test gases. This method and accompanying results will be presented in detail to illustrate benefits of the novel assembly.
Monday
Single use membrane adsorbers with high productivity for bioseparations
05:50pm - 06:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Nonwoven-based membrane adsorbers could have an important impact on current bioprocess operations by providing an alternative single-use disposable product capture step with a significantly higher productivity than chromatography columns. To this aim, poly(butylene) terephthalate (PBT) nonwoven fabrics were UV grafted with a glycidyl methacrylate (GMA) layer that allows functionalization with different ligands. With cation and anion exchange groups, it was possible to measure dynamic binding capacities (DBCs) at very short residence times (0.1 – 0.5 minutes) that are similar or higher than those obtained using the best ion exchange chromatography resin columns with much longer residence times. For example, sulfonated UV grafted PBT-GMA-SO3 cation exchange membranes exhibit a DBC10% of 181.72 mg IgG/mL at a residence time of 0.5 minutes, and a DBC10% of 106 mg IgG/mL at 0.1 minutes residence time. These membranes also resulted in high yields (>97%) and purities of IgG from CHO cell culture supernatants.

We also present results using a GMA-grafted PBT nonwoven fabric modified by attachment of iminodiacetic acid (IDA) as a cation exchange membrane. The optimized membrane exhibits excellent flow permeability and achieved a DBC10% of 76.6 mg/mL for spiked hIgG from CHO cell culture at 0.1 min residence time, higher than most cation exchange resins. The membranes were also tested using a CHO culture supernatant with a single-chain variable fragment (with no Fc domain) and achieved a binding capacity of about 150 mg/mL with good clearance of HCP and DNA. Because of their ability to work at shorter residence times, these membranes have much higher productivity that columns, and provide a disposable, high-throughput, high capacity alternative to chromatographic resins for the production of mAbs and other biotherapeutics.

Monday
Rapid, high-throughput, mRNA purification: Preparing for tomorrow
06:10pm - 06:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 05
Dr. Graham Temples, Presenter, Purilogics; Jinxiang Zhou; Bin Guo
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
FDA Emergency Use Authorization of mRNA-based COVID-19 vaccines is a critical milestone in mRNA medicines and foreshadow the high growth potential of mRNA drugs. However, CEO of a leading pharmaceutical mRNA company suggested the quantity and quality, i.e. consistent purity, of mRNA remain as bottlenecks for their production. Particularly, the lack of high-throughput downstream purification processes is a major challenge in the upscaling of industrial mRNA production. There are few commercially available mRNA affinity purification tools, especially for process scale operations. Purilogics is presenting a novel mRNA affinity membrane designed for rapid purifications. In this presentation, we will compare the effect of flowrate and mRNA size on the binding capacity and recovery of our membrane product against the current gold standard commercial mRNA affinity resin product. Additionally, purifications assessing purity and yield from industrially relevant in vitro transcription reactions will be demonstrated.
Biomedical Technologies: Imaging, Diagnostics, & Other Integrative Approaches to Study & Model Diseases: Biosensors & Probes for Medical Diagnostics
04:30pm - 06:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Yonghyun Kim, Organizer, The University of Alabama; Maryam Raeeszadeh Sarmazdeh, Organizer, University of Nevada, Reno; Adam Melvin, Presider, Louisiana State University; Divya Chandra, Presider, ‍ ; Christopher Canova, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

A comprehensive understanding of diseases coupled with their effective diagnosis and treatment is inherently linked to the technologies available to doctors, clinicians, and researchers. With the advent of personalized medicine, many times bioanalytical and biochemical approaches to study and treat diseases are limited by the available technology. This has led to new approaches to perform (i) high-throughput single cell analysis, (ii) point-of-care detection of biomolecules, viruses, and cells, (iii) large scale analysis and processing of massive data sets, and (iv) low volume detection of disease biomarkers. Researchers have harnessed a range of tools and approaches including nanoparticles, microfluidics, tissue engineering, peptides, proteins, DNA, RNA, 3D printing, microscopy, and spectroscopy to name a few. This has led to selective and sensitive biosensors with low limits of detection and high signal-to-noise ratios that can be incorporated into high-throughput or field ready approaches to rapidly analyze biological samples. Recent years have also produced novel in vitro models that better recapitulate the in vivo environment to increase the fundamental understanding of disease progression. This has led to new approaches in cell culture co-culture and 3D culture that incorporate more realistic biomaterials to better represent tissue.

Monday
Elucidating neurochemical communication via DNA aptamer-modified nanopores
04:30pm - 04:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Advancing our understanding of neuronal communication necessitates novel nanotools that can monitor chemical signal transduction in close proximity to nanoscale synapses. While electrophysiology platforms such as microelectrode arrays are well-established to record electrical signaling from neurons in vitro, the translation of methods that can monitor chemical signaling from neurons have been inadequate. We have tackled this challenge by coupling the inherent selectivity of DNA-based recognition elements termed aptamers, with nanoscale pipettes with openings of ca. 10 nm. Aptamers are systematically designed oligonucleotide receptors that exhibit highly specific and selective recognition of targets. Aptamers that recognize small-molecule neurotransmitters have recently been isolated. Upon reversible target binding, aptamers undergo a rearrangement of the negatively charged backbone and these dynamic structural changes can be transduced as measurable changes in current through the nanoscale orifice of the probes. In conically shaped nanopipettes with pore diameters of comparable size to the electrical double layer, the ion current rectification effect is observed. This phenomenon manifests as asymmetric ionic flux through the nanopores, dependent on the surface charge that is modulated by the aptamer rearrangement upon target recognition. Nanoscale confinement of ion fluxes, analyte-specific changes in molecular conformation of aptamer species, and related surface charge variations enabled highly sensitive detection of neurotransmitters, even in complex environments. We demonstrated the capacity to detect physiologically relevant differences in neurotransmitter amounts in media collected from human induced pluripotent stem cell-derived neuron cultures with unprecedented sensitivity. In the future, we aim at integrating aptamer-modified nanopipettes as probes to scanning ion-conductance microscopy to facilitate real-time sensing from live neurons.

Monday
Split fluorescent light-up aptamer probes for label-free pathogen detection
04:50pm - 05:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Ryan Connelly; Jack Mordeson; Pedro Madalozzo; Dr. Yulia Gerasimova, Presenter, University of Central Florida
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Nucleic acid-based assays allow for rapid detection of bacterial and viral pathogens and sequence-specific analysis of their genetic signatures. In this respect, hybridization probes are important tools to ensure specificity of the analysis. Fluorescent light up aptamers (FLAPs), which bind low-fluorescent dye ligands and increase their fluorescence, offer an advantage of label-free signal reporting. Therefore, FLAP-based hybridization probes are promising for molecular diagnostics.
Recently, a dapoxyl dye-binding FLAP has been reported. We have demonstrated that the same aptamer sequence can also bind and enhance fluorescence of other fluorogenic dyes including commercially available arylmethane dyes. Such binding promiscuity allows one to design an aptasensor with multiwavelength reporting capabilities depending on the dye used. We have designed a split dapoxyl aptamer (SDA) probe and applied it to the detection of the amplified fragments of bacterial and/or viral genomes obtained using nucleic acid sequence-based amplification (NASBA). Four different fluorogenic dyes were explored with the SDA probe, which can be designed do differentiate the nucleic acid targets down to single-nucleotide substitutions. The linear dynamic range and limit of detection are not significantly affected by the changes in the dye used. The signal can be conveniently observed by the unaided eye when excited with a portable light source. The system is tolerant to potential inhibitors from the amplification reaction, so that the amplicon can be interrogated by the probe directly in the amplification sample without the need of the amplicon isolation. This opens a possibility to use the SDA probe in a one-tube format, where all the components for the amplification and detection reactions are added simultaneously. We have also discovered that nucleotide substitutions at specific positions of the dye-binding core of the aptamer affect its ability to trigger fluorescence of some dyes, but not the others. This property can be used to design a set of aptasensors for multiplex target analysis using one and the same aptamer as a scaffold.

Monday
Point-of-care microchip electrophoresis for integrated anemia and hemoglobin variant testing
05:10pm - 05:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Ran An, Presenter, Case Western Reserve University; Umut Gurkan
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Anemia affects 25% of the world's population with the heaviest burden borne by women and children. Genetic hemoglobin (Hb) disorders, such as sickle cell disease (SCD), can independently induce hemolytic anemia causing high morbidity and mortality. Anemia and SCD are inherently associated and are both prevalent in low-resource settings thus require integrated point-of-care (POC) detection for both diseases.
Electrophoresis has been listed by the World Health Organization as an in vitro diagnostic technology for Hb testing. Here, we developed a paper-based microchip electrophoresis system to allow 2-steps Hb separation within single test (Fig. 1A). The first step separation enables separation between total Hb and a standard calibrator. The second step separation enables separation among various Hb variants (Fig. 1B&C). Implementing machine learning algorithm, migration patterns and relative abundancy of Hb band and standard calibrator band are recognized for Hb level determination and anemia detection. Hb variants are identified according to the final migration distances and quantified for identification of Hb disorders such as SCD.
46 clinical subjects were tested in this study. Hb levels determined using the reported technology demonstrated high association with Pearson Correlation Coefficient of 0.95 compared to the results of standard reference complete blood count (Fig.1D) and high reproducibly with a mean absolute error of 0.55 g/dL and a bias of -0.10 g/dL (95% limits of agreement: 1.5 g/dL) according to Bland-Altman analysis (Fig. E). Anemia determination was achieved with 100% sensitivity and 92.3% specificity with a receiver operating characteristic area under the curve (AUC) of 0.99 (Fig.1F). With the same test, subjects with sickle cell disease were identified with 100% sensitivity and specificity.
Overall, we demonstrate a technology to enable, for the first time, reproducible, accurate, and integrated blood Hb level prediction, anemia detection, and Hb variant identification in single test at the point-of-care.

Monday
Detection of salivary 1,5-anhydroglucitol for convenient, non-invasive diabetes mellitus screening
05:30pm - 05:50pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Diabetes mellitus is a severe, chronic disease that affects over 420 million people worldwide. If left untreated, diabetes can lead to severe complications and death. The onset of complications can be delayed or even prevented when the disease is diagnosed early, but it is estimated that nearly 40% of all persons with diabetes are undiagnosed. Screening in communities with limited access to healthcare can significantly reduce the number of undiagnosed cases of diabetes. The blood concentration of 1,5-anhydroglucitol (AHG), a naturally-occurring six-carbon monosaccharide similar in structure to glucose, falls during periods of hyperglycemia as glucose outcompetes AHG for kidney reuptake. Blood AHG measurement is an FDA-cleared method of monitoring glycemic control, and salivary AHG has been suggested to be useful for diabetes screening. However, previous efforts to measure AHG in saliva have been unsuccessful. We have developed a chemiluminescence assay to quantify AHG in saliva and demonstrated that the assay could distinguish between healthy and treated-diabetic individuals (N=265; p < 0.001, ROC AUC 0.82). These results suggest that, with further validation, this approach may serve as the basis of a non-invasive tool to screen for diabetes.
Monday
Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout
05:50pm - 06:10pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems Nucleic Acid Sequence Based Amplification (NASBA) is one well-known example. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.

Monday
Withdrawn
06:10pm - 06:30pm USA / Canada - Eastern - August 23, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual