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COVID-19 Diagnostics & Detection - Technologies, Challenges & the Scale-Up of Testing: COVID-19 Diagnostics & Detection - Technologies, Challenges & the Scale-Up of Testing
07:00pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Frank Kotch, Organizer, Pfizer Inc; Varnika Roy, Organizer, GlaxoSmithKline Plc; Dr. Shannon L Servoss, Organizer, University of Arkansas; Catherine Fromen, Presider, University of Delaware College of Engineering; Yu-Shan Lin, Presider, Tufts University; Robert Pantazes, Presider
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Co-sponsor/Theme: Theme: Resilience of Chemistry
Division/Committee: [BIOT] Division of Biochemical Technology

As the COVID-19 pandemic spread across the globe, it quickly became clear that detection of SARS-CoV-2 infection is a key necessity in fighting viral spread. As such, numerous detection methods, both established and novel, were implemented for the detection of current and past SARS-CoV-2 infections. This session will focus on the development of virus and antibody detection methods, as well as other COVID-19 diagnostics. Challenges on speed for commercialization including but not limited to regulatory hurdles to get these novel technologies to patients faster in a pandemic situation would be discussed.

Tuesday
Multiplex and colorimetric assay of SARS-CoV-2, influenza A and B viruses using point-of-care toolkit
07:00pm - 07:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Dohwan Lee, Presenter, Georgia Institute of Technology; Chia-Heng Chu; A. Fatih Sarioglu
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Since the first case of a novel coronavirus disease (COVID-19) in Wuhan, China at the end of 2019, the causative agent named severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) has been spreading around the world causing immense public health and socioeconomic problems. This tremendous spread of the COVID-19 is attributed to the fact that the disease can often be asymptomatic or its symptoms can be misinterpreted as due to the flu. To enable timely response and effectively prevent the virus transmission, frequent self-testing methods that can specifically identify SARS-CoV-2 by distinguishing from influenza viruses are needed. However, the current diagnostic methods (e.g., RT-qPCR) demands a bulky and expensive thermal cycler with a prolonged assay time and painstaking steps by trained technicians with sophisticated laboratory instruments, which limits their use in point-of-care (POC) settings. In this work, we report a fully-integrated POC toolkit for multiplex molecular diagnosis of SARS-CoV-2, influenza A and B viruses from saliva. The paper-based device was physically programmed with a built-in capillary flow regulation by creating integrated timers on flow paths. The sequential routing of the capillary flow on paper with controlled delays enables to run a sequence of chemical reactions and internally generate heat to drive those reactions to autonomously extract, amplify, and detect the viral RNA. Using our assay, we could identify SARS-CoV-2 and influenza A and B viruses at concentrations as low as 5 copies/μL visually from a colorimetric analysis. The capability to autonomously perform a traditionally labor-intensive genetic assay on a disposable platform will enable frequent, on demand self-testing, a critical need for POC testing to positively identify COVID-19 infections.
Tuesday
Electronic enzyme linked immunosorbent assay for detection of COVID19 biomarkers
07:20pm - 07:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Neda Rafat, Presenter, Georgia Institute of Technology; Aniruddh Sarkar; Hanhao Zhang; Preetham Peddireddy
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The unprecedented global spread of COVID-19 and its symptomatic heterogeneity have underscored the need for developing accurate yet inexpensive diagnostic and monitoring tools for infectious diseases. Enzyme-Linked Immunosorbent Assays (ELISA), widely used for sensitive measurement of various biomarkers, are currently limited to complex, expensive, and bulky lab-based instruments.

We report an inexpensive electronic ELISA (E2LISA) which enables direct electrical detection of molecular biomarkers from a single drop of sample via probe-directed, enzymatically amplified metal nanoparticle deposition on microelectrodes. Capture agents, such as antigens, are coated on interdigitated gold microelectrodes on a glass microchip, and then exposed to sample to selectively bind the analyte to which a horseradish peroxidase (HRP) enzyme-labeled probe solution is then applied. Addition of a substrate results in deposition of silver nanoparticles (Fig. 1A). The conductive layer bridging the electrode gap enables the passage of current upon application of a voltage providing a direct electronic readout.

Here we first used a model binding reaction of biotin-labeled bovine serum albumin and streptavidin-HRP to optimize E2LISA performance across various chip design and experimental parameters such as the electrode gap, substrate concentration, reaction time, and impedance measurement frequency. Additionally, we observed that a more uniform metal layer and thus a more robust and repeatable electronic signal could be generated by the addition of probe-labeled gold nanoparticles to the immunoassay.

Detection of SARS-CoV-2 spike protein-specific IgG antibodies, which are a biomarker of current or prior infection with the virus, was demonstrated from convalescent COVID19 patient serum (Fig. 1B). All patient samples showed high silver deposition and low measured microelectrode impedance (Fig. 1C). Healthy human serum showed little or no silver deposition and hence a high measured electrode impedance. Further development will include the detection of viral antigens and nucleic acids, which are an early biomarker of infection, using appropriate capture agents and probes.

Tuesday
One of the crucial steps in deploying plasma therapy for COVID-19 is the development of critical assays, including ELISA, to detect SARS-CoV-2 antibodies and evaluate their potential suitability. Unfortunately, some assays for assessing neutralizing antibodies involve using live SARS-CoV-2 virus in a high containment facility (BSL3) and can take up to a week to complete. The assay cost, time for completion, and requirement for PPE make it a weak link in the pipeline for identifying plasma donors. Current commercial ELISAs provide primarily a yes/no answer and lack the precise quantitation of protective antibody levels desired in sourcing plasma. Therefore, the goal of this project is to biomanufacture and rapidly deploy coronavirus antigens in order to scale up a quantitative ELISA assay for characterizing plasma needed for treatment and prophylaxis. Our team is developing methodologies to generate purified SARS-CoV spike (S) protein and the SARS-CoV-2 S protein receptor binding domain (S-RBD) to be incorporated into the quantitative ELISA assay. The presence of an extremely sensitive and robust ELISA assay using multiple antigens will specify the highest binding and protective antibodies (and their targets) from a pool of volunteers and recipients. Additionally, the team is collaborating to develop an automated ELISA robotic system to increase efficiency and speed, ensuring reproducibility when performing candidate screening. This presentation will discuss the production of multiple S and RBD protein reagents using various production and purification platforms and the evaluation of their effectiveness through quantitative ELISA kits.
Tuesday
Optimization of biosensing platform for the ultrasensitive quantification of SARS-CoV-2 specific IgG antibody response post-infection
08:00pm - 08:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Herein, we report the fabrication of an ultrasensitive, multiplexing, high-throughput nanoplasmonic-based biosensing platform for the detection of antibody response to SARS-CoV-2. The biosensing device was fabricated utilizing gold triangular nanoprism (Au TNP) functionalized glass coverslips in a multi-well format. Previously, we have shown that this biosensing device is capable of detecting IgG antibodies utilizing the localized surface plasmon resonance (LSPR) property of the Au TNPs at a limit of detection in the high aM range. However, an ultrasensitive biosensing platform with limit of detection in the low aM to zM range is required in order to quantify and differentiate the antibody levels in response to SARS-CoV-2 at different stages of post-symptom onset. Quantifying the antibody levels at different stages is important to determine whether the antibody levels persist or alternatively decay over time, as well as to determine how long they are present in human biofluids. To achieve ultra-sensitivity of our LSPR-based biosensors, we optimized the structural parameters through (1) control manipulation of receptor (SARS-CoV-2 spike glycoprotein subunit 1 and a specific peptide sequence), and (2) the geometric dimension of Au TNPs. These optimization steps provided an ultrasensitive biosensing platform capable of assaying SARS-CoV-2 specific IgG with a limit of detection of high zM. As a proof of concept, we utilized the biosensing platform to quantify the level of IgG in a cohort of 100 post-infected COVID-19 patients and determined the antibody levels at different stages of infection (post 10 days, 20 days, 30 days), overall showing a stable IgG level for this time period. Taken together, we believe this ultrasensitive biosensor platform will provide a way to quantify the level of antibodies present post infection of not only COVID-19, but also other viral infections.
Tuesday
The COVID-19 pandemic has highlighted the importance of accessible and affordable diagnostics to control the spread of infectious diseases. Some critical bottlenecks to widespread population testing include risks to healthcare workers administering tests, shortages of PPE, and cold-chain requirements to transport biological specimens or reagents for tests. To overcome these limitations, we have developed a paper-based SARS-CoV-2 diagnostic that does not require a cold-chain and employs self-testing and wireless detection in a closed envelope to prevent exposure to healthcare workers and eliminate the need for PPE. The electrical transducer is a wireless, battery-free LC resonant sensor that can be interrogated through non-metallic materials. This passive LC circuit is screen-printed on paper and coated with gelatin switches that, upon degradation, shift the resonant frequency of the sensor. To make this selective to SARS-CoV-2 genome targets, the expression of a gelatin-degrading catalyst is regulated by a toehold switch. In the presence of SARS-CoV-2 RNA, the toehold switch relaxes and subtilisin is expressed, thereby degrading the gelatin substrate. A novel resonator design utilizing electromagnetic active and dead zones increases sensitivity and reduces by 100-fold the working sample volume of analyte from previous coating degradation work, enabling use of volumes down to 10 µL. Cell-free protein synthesis of reporter enzyme is done using custom cell extract replacing the need for expensive commercial in vitro kits which drastically reduces the price point of the sensors. Also, lyophilization of cell extract in the paper-based device removes the need for a cold-chain and allows for stockpiling in advance of future pandemics. Exposed sensors can be rapidly scanned after being mailed from three different laboratories which demonstrates proof of concept of mail-in approach. This work presents a novel paper-based, inexpensive, modular sensing platform which can be applied to the detection of many other endemic and emerging infectious diseases.
Tuesday
Rapid and Inexpensive point-of-use (POU) testing for SARS-CoV-2 quantification with membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system
08:40pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population and provide early detection of contamination via wastewater-based epidemiology (WBE). But widespread WBE for SARS-CoV-2 quantification would rely on the availability of specialized equipment and personnel for environmental (i.e., wastewater) sample preparation, processing, and analysis that are currently prioritized to meet the demand for clinical samples analyses. Here we demonstrated the usage of our portable membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system for absolute quantification of SARS-CoV-2 in wastewater samples within a 1h-timeframe for point-of-use (POU) testing and data management, which was compared with the performance of golden standard reverse-transcription quantitative polymerase chain reaction (RT-qPCR) method. The limit of detection (LOD) of mgLAMP for SARS-CoV-2 quantification in Milli-Q water was observed to be down to 1 copies/mL, and that in surface water collected from Kathmandu, Nepal was down to 50 copies/mL. Both were 100-fold lower than that of RT-qPCR in corresponding matrices. A 3D-printed portable device integrating incubation and illumination was manufactured to simultaneously allow the POU operation and analysis of 9 mgLAMP assays. Quantitative result of the virus concentration can be sent back to a smart phone or stored in an online database. Compared to alternative detection methods, our platform has a very high level of tolerance against inhibitors due to the restriction effect of the hydrogel matrix, which allows for the highly sensitive detection in either clinical samples or environmental samples. Additional merits of our detection platforms are portability, cost-effectiveness, user-friendliness and versatility, allowing for water environmental and clinical POU testing applications in low-resource settings.
Big Data in Discovery & Development of Biopharmaceuticals: Big Data in Discovery & Development of Biopharmaceuticals
10:30am - 12:30pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 06
Sandeep Kumar, Organizer; James Lalonde, Organizer, Codexis Inc; Richard Fox, Presider, ‍ ; Hector Garcia-Martin, Presider, ‍ ; Iman Farasat, Presider, ‍ ; Stanley Krystek, Presider, Bristol-Myers Squibb
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This session will describe different uses of big data and analytical tools such as ML and AI to guide protein engineering, design and selection efforts aimed at improved developability (manufacturability, safety, efficacy and pharmacology) of biopharmaceuticals.

Wednesday
Engineering novel proteins: Protein design, directed evolution, and machine learning
10:30am - 11:10am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 06
Grant Murphy, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Enzymes are exceptional catalysts with unparalleled selectivity and rate enhancement. In many cases, enzymes also offer significant advantages over traditional catalysts with respect to cost, safety, and environmental impact. Due to these properties, the use of enzymes as biocatalysts in industrial settings has grown dramatically. Central to this growth has been the ability to create novel enzyme functions and properties using protein design and engineering. The fields of biocatalysis and protein engineering have matured to the point where the identification of new enzyme activity and further engineering of an enzyme for industrial purposes has become almost routine. These advancements have made it practical to consider moving beyond the engineering of a single enzyme and to consider the invention of entirely novel multi-enzyme cascades not found in nature to synthesize compounds not found in nature. These novel pathways could be considered ex vivo metabolic processes; enzyme cascades created and occurring outside the confines of living systems. This talk will highlight several current examples of our efforts to engineer novel enzyme cascades to synthesize Merck compounds from simple starting materials. The talk will cover our bioinformatic and design approach, our engineering process, and our machine learning efforts.
Wednesday
Cell-free prototyping and rapid optimization of biosynthetic enzymes for cellular design
11:10am - 11:30am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 06
Michael Jewett, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts, and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. In this presentation, I will describe the use of cell-free systems coupled to machine learning for optimizing the biosynthesis of 3-hydroxybutyrate, acetone, and products from reverse beta oxidation. We then show how iPROBE selected pathways can be used in Clostridium autoethanogenum to produce bioproducts solely from carbon waste gas. We expect iPROBE coupled to machine learning to accelerate design–build–test cycles for industrial biotechnology.
Wednesday
MSA transformer
11:30am - 11:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Unsupervised protein language models trained across millions of diverse sequences learn structure and function of proteins. Protein language models studied to date have been trained to perform inference from individual sequences. The longstanding approach in computational biology has been to make inferences from a family of evolutionarily related sequences by fitting a model to each family independently. In this work we combine the two paradigms. We introduce a protein language model which takes as input a set of sequences in the form of a multiple sequence alignment. The model interleaves row and column attention across the input sequences and is trained with a variant of the masked language modeling objective across many protein families. The performance of the model surpasses current state-of-the-art unsupervised structure learning methods by a wide margin, with far greater parameter efficiency than prior state-of-the-art protein language models.
Wednesday
Using deep learning and MD simulations to model protein interactions
11:50am - 12:10pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 06
Imee Sinha, Presenter, Rensselaer Polytechnic Institute; Dr. Camille Bilodeau, Massachusetts Institute of Technology; Shekhar Garde; Steven Cramer, Rensselaer Polytechnic Inst
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Fundamental understanding of water-mediated biomolecular interactions is one of the most challenging problems in science. Studies of biomolecular interactions using molecular dynamics simulations or experiments (e.g., atomic force microscope or other molecular tools) are extremely time consuming and costly. Recently it was shown that quantifying the collective properties of water near protein surfaces, specifically, the free energy of dehydration of surface patches using indirect umbrella sampling (INDUS) simulation technique, contains key information about biomolecular interactions. However, despite the efficiency of INDUS, it is not feasible to sample the vast space of surfaces of all proteins. We believe that a new approach based on AI informed by data from selected INDUS simulations can provide the solution. In this work, we employed sparse INDUS sampling to generate dehydration free energies for a limited number of protein patches. This data was then used to augment a semi supervised variational autoencoder which maps structural and chemical features of protein patches to their hydrophobicity. Our deep learning model can predict context dependent hydrophobicity for protein patches that is several orders faster than INDUS or other techniques. We also observed fascinating insights on how contextual environment and topography shapes the hydrophobicity of protein surface patches. Further, this work can allow users to place a “quilt” on the protein of interest, dividing the surface into patches based on hydrophobicity, and using that information to predicting interaction of proteins with partners of interest.
Wednesday
Machine learning methods for Pareto optimal antibody engineering
12:10pm - 12:30pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Therapeutic antibody development requires precise co-optimization of myriad biophysical properties, many of which exhibit strict tradeoffs during protein engineering campaigns. One such tradeoff is often seen between arguably the two most important antibody properties: affinity and specificity. Co-optimization of these two properties remains challenging due to experimental limitations and obstacles associated with developing robust models that accurately predict analog binding values from deep sequencing datasets. We have sought to address these challenges by developing a comprehensive methodology for the acquisition of high-quality deep sequencing data and subsequent analysis with machine learning models. We have compared several sequence-based feature extraction methods for analysis via machine learning models, achieving accurate predictions of analog binding strengths from binary sequencing labels. This enables tunable Pareto optimization of antibody properties from high-throughput experimentation, which greatly reduces the amount of experimentation needed for co-optimizing key properties for generating potent antibody therapeutics.
Process Development and Reactor Engineering: Process Development and Reactor Engineering
10:30am - 12:30pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 03
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Bruce Levine, Presider, ‍ ; Krishanu Mathur, Presider, ‍ ; Huong Le, Presider, Amgen Inc.
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Technological advances in the approaches and methods for ex vivo cell, tissue, and gene manipulation have led to investigations of cells, gene modified cells, and tissues endowed with enhanced potency and unique functions, with promise of a new generation of infused therapeutics. Cell-based therapies began with experimental blood transfusions and bone marrow or hematopoietic stem cell transplants. With greater understanding of the biology, new technologies have evolved so that a new pillar of medicine is now being created. Translation of research findings to a final drug product requires strategic merging of science and technology with emphasis on safety, purity, potency, and identity of the product. This session will encompass multiple aspects of cell therapy process development where the cells, gene modified cells, gene edited cells, or cell derived vectors which encode the gene of interest are the products intended for investigation in humans or veterinary applications. This would include advances in the isolation, culture, gene transfer and modification, process scale up, culture medium design, testing, and characterization of cell-based therapy products. Papers relevant to these topics are highly encouraged, including those focusing on novel process improvements, control and optimization strategies, equipment and reagents design and characterization.

Wednesday
Rapid CHO cell process intensification for mAb production: Implementation of an intensified fed-batch with doubled space-time yield
10:30am - 10:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
During the last decade many different mammalian upstream process strategies emerged in the field of process intensification (PI). These offer the potential to optimize the productivity and reduce plant’s footprints, ultimatively increasing flexibility and lowering expenditures.
Many of this novel strategies utilize perfusion cultivations to either generate high cell concentrations (>100x106 cells/mL) during N-1 stage for the inoculation of subsequent (intensified) fed-batches (FB) or for the long-term N-stage production of biopharmaceuticals. Furthermore, related and elaborated processes were developed, such as using N-1 perfusion as continuous cell supply for an N-stage chemostat or concentrated FBs, which are perfusion processes with product retention.

Despite the advantages of PI, their implementation into established process platforms still requires certain preceding investigations to identify and evaluate the intrinsic optimization potential. Influencing factors represent seeding ratios, feed timing and proportions, potentially even adaptations, and also process parameters (pH, T, DO,…). All of them finally contribute to key process indicators (KPI), the most prominent ones being product titer and quality, which are a consequence of cell growth and metabolism under the respective conditions. In order to allow for a rapid integration of PI, it is crucial to conduct initial investigations in a fast and reliable manner.

In this talk, we show how a conventional FB platform process (15 mL – 2000 L) was transitioned rapidly into an intensified process using multi-parallel small-scale bioreactor systems. Experiments were conducted consecutively in 15, 250 mL and 5 L bioreactors and allowed to evaluate the impact on KPIs: the space-time yield (STY) was successfully doubled, while the product quality was maintained. Potential impacts of PI on cell metabolism were evaluated using flux balance analysis and found to fade out towards the process’ end.

This work intends to further challenge both academia and industry to deploy upstream PI potentials in the future.

Wednesday
Reproducing dynamic environment in microfluidic single-cell cultivation based on computational lifeline analysis
10:50am - 11:10am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The biotechnological production of valuable substances is typically complicated by the loss of microbial performance upon scale-up [1-3]. This challenge is mainly caused by discrepancies between homogeneous environmental conditions at laboratory scale, where organisms are optimized, and inhomogeneous conditions in large-scale bioreactors, where the production takes place. To improve strain selection and process development, it is thus of major interest to characterize these fluctuating conditions at large scales and investigate their impact on microbial cells.

In this contribution, we will demonstrate the high potential of dynamic microfluidic single-cell cultivation combined with computational fluid dynamics (CFD) simulation of large-scale bioreactors. CFD simulations of a 300 L bioreactor were applied to characterize environmental conditions in large-scale bioreactors. So-called lifelines were determined by simulating multiphase turbulent flow and mass transport combined with particle tracing. Glucose availability experienced by the microorganism Corynebacterium glutamicum was traced. Resulting lifelines were discretized into low, medium and high glucose availability regimes. Discretized lifelines were used as feeding profiles of a dynamic microfluidic single-cell cultivation (dMSCC) system to investigate how the fluctuating glucose concentration affects cellular physiology and colony growth rate.

The presented approach paves the way for an improved understanding of how the cellular lifelines of large-scale bioreactors influence the cellular response within growth and production. It also provides insights into how to understand the conditions in large-scale bioreactors from the view of a microorganism and the dependence of cell wellbeing on the observed conditions.

Wednesday
High CHO cell density harvest clarification: Acidic pre-treatment followed by Clarisolve® depth filters exhibits multiple benefits such as filtration area reduction and filtrate quality improvement
11:10am - 11:30am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Current evolution to high cell density and high product titer monoclonal antibody CHO cell cultures is placing a larger burden on traditional downstream clarification and purification operations.To alleviate this challenge, various types of pre-treatments are being employed to make the clarification process more efficient. Pre-treatment is accomplished either through the reduction in pH of the feedstream (acid precipitation) or through the addition of cationic polymers (flocculation). These methods result in aggregation of solid and soluble particle impurities into larger agglomerates, thus reducing small particles which are difficult to centrifuge and can plug downstream filters. In this study we implement and demonstrate the benefits of combining high CHO cell density harvest with acid pre-treatment followed by Clarisolve® depth filters. Clarisolve® depth filters are specifically designed to handle very large particles sizes. Alternatively, Millistak+® depth filters were assessed without any harvest pre-treatment. The results show that acidic pre-treatment followed by Clarisolve® depth filter enhanced primary depth filtration performance as compared to traditional calrification with no pre-treatment. Also this method was able to reduce impurities in filtrate significantly to make less loading on further downstream processing.
Wednesday
Novel continuous bioreactor for increased cell production in upstream bioprocessing
11:30am - 11:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 03
Rithvija Avvari, Presenter, Auburn University; Thomas Hanley; Paul Todd
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Conventional bioreactors employ impellers and do not offer low shear environment for cell sustainability. Also, consistent cell production and quality are difficult to achieve in these reactors. With improved continuous biomanufacturing techniques, these issues can be addressed. A continuous bioreactor with spiroid was prototyped using 3D printing techniques. The bioreactor is a horizontal cylinder rotated on a roller bed. The spiroid is embedded in the cylindrical wall of the bioreactor to increase oxygen transfer to the liquid phase which fills two-thirds of the bioreactor. The rotational rate of the reactor can be adjusted to control the flow of gas and liquid in the spiroid. When the partially filled reactor is rotating, the spiroid picks up slugs of gas and liquid near the reactor exit and delivers them to the reactor entrance. Thus, the spiroid substitutes for a traditional impeller to increase mixing and oxygen transfer without generating higher shear forces. This bioreactor can be operated in either batch or continuous modes with inlet flows via rotary unions available to provide medium and oxygen and outlet flows for waste and in-line analysis. Computational fluid dynamic studies showed that the spiroid increases oxygen transfer at higher rotation rates of the reactor. Cell production increase was validated by culturing Saccharomyces cerevisiae in both batch and continuous modes with and without spiroid. Cell growth was monitored at different operating conditions using a spectrophotometer. The reactor with spiroid showed an increase in cell production and decrease in operating time. Steady-state cell concentration was achieved for different flow rates in the reactor. Overall, the spiroid in this continuous bioreactor shows a promising approach in enhancing cell production while maintaining consistent product quality.
Bioreactor Schematic

Bioreactor Schematic


Wednesday
This work describes the combined use of high throughput technologies and novel applications of process capability methodology to drive efficiency and robustness in biologics process development and process characterization. Process capability is commonly measured by Ppk; however, it is typically not evaluated until a sufficient number of commercial lots have been executed at which point there may be limited opportunity to make process adjustments to enhance robustness. This work presents a case study in which prior knowledge and SME input have been utilized to assess process capability during development to assess likelihood of long-term robustness, establish readiness for scale-up/technology transfer, and identify focus areas for process characterization. This work also discusses implementation of the high throughput Ambr250 bioreactor system with automated sampling for process development and characterization which has driven significant efficiencies in execution time and cost. The combination of high throughput technologies and novel application of process capability methodology can be used together to expedite early insight into potential process vulnerabilities, provide a common platform to understand risk and inform decision making, and provide clear targets against which to measure process success, ultimately resulting in higher process knowledge and process robustness at launch.
Wednesday
High throughput scale-down harvest method to support upstream process development and characterization efforts using mini bioreactors for IgM antibodies
12:10pm - 12:30pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Mammalian cells are routinely used to produce antibodies and other recombinant biologics. Increasing demands for biologics and the need to reduce time to patients has created the need to adopt high throughput systems to speed up development. We have evaluated automated mini bioreactors such as the ambr® 250 as a potential scale-down model for upstream process development in the manufacture of IgM antibodies. These mini bioreactors offer many advantages, but also some drawbacks. For example, harvesting low cell culture volumes poses some challenges. Using a standard two-step depth filter operation can result in lower recovery, whereas centrifugation alone is inefficient in removing host cell protein (HCP) and DNA from the harvested cell culture fluid (HCCF). This results in an HCCF impurities profile that is not suitably comparable to pilot scale or large scale, thus interfering with the scaling of initial purification steps.

To address these issues, we developed a scale-down harvest method that delivers IgM material with product quality attributes comparable to large-scale harvest. Our scale-down harvest method consists of a two-stages, centrifugation followed by secondary depth filtration, which serves as a polishing step and aids in reducing process impurities. This method was evaluated for process yield as well as HCP and DNA removal. This scale-down harvest method has the potential to reduce both material and time requirements for process development and characterization studies. We will present screening studies evaluating commercially available secondary depth filters and their performance in efficiently removing process impurities in post centrifuged cell culture fluid, thus improving recovery of IgM antibodies.

Advances in Process Analytical Technologies for Measurement & Control: Advances in Process Analytical Technologies for Measurement & Control
10:30am - 11:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 04
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Jennifer Pollard, Presider; Steven Evans, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This session will focus on process analytic technologies (PAT) that enable real-time control, faster batch release, or improved process understanding for downstream processing. The development and implementation of in-line and/or at-line process analytics may include sensors based on, for example, Raman or infrared spectroscopy, particle size imaging, variable pathlength flow concentration measurement technologies or other technologies. In this session, we’d like to explore the identification of innovative tools or a novel implementation of established methods in downstream manufacturing processes for one or more downstream unit operations. Case studies showing how these tools can enable meeting in-process limits, with the goal of meeting a set of final critical quality attribute (CQA) limits, or how they provide a more in depth understanding of the process are encouraged. Any challenges with the implementation of such PAT solutions and their impact on the overall control strategy may be presented. Topics may include implementation of integrated control strategies that leverage enhanced product understanding for manufacturing process optimization, process characterization, or regulatory submissions. Additionally, leveraging PAT to enable understanding of downstream process capabilities and their impact on CQAs are among topics of interest.

Wednesday
Withdrawn
10:30am - 10:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Wednesday
Development of Quartz Crystal Microbalance Arrays for Analysis of Complex Biomanufacturing Medias
10:50am - 11:10am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 04
Bang Hyun Lee; Travis McKay; Srivatsan Ramesh, North Carolina State University; Stefano Menegatti; Dr. Michael Daniele, Presenter, NC State University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Conventional biomanufacturing process analytical technologies (PATs) are limited by costly and time-consuming offline efforts, which require extensive bioseparation and sample handling. With the evolution of therapeutics towards biologics development over the years, it is more relevant than ever to create scalable, in-line, and high throughput quantification methods to interrogate complex cell culture media and lysates directly. Specifically, it is essential to quantify the rate of target production, such as host cell proteins (HCPs). A quartz crystal microbalance (QCM) biosensor was integrated with microfluidic dilution generators to demonstrate the detection of HCPs. Surface chemistry was optimized using thiolated polyethylene glycol (PEG) as a unit for the self-assembled monolayer (SAM) with IgG-binding peptide as a capturing moiety. Spectroscopic ellipsometry was employed to characterize the surface chemistries by non-destructively analyzing the dielectric properties of thin films. In addition, atomic force microscopy images have been produced to probe the local structural analysis of SAM on gold surfaces. Comparing the various methods of immobilizing the PEG-peptide complex on the gold surface led to crafting the proper protocol implemented on the QCM sensor for further assays. The custom QCM biosensor platform was validated by measuring the adsorption and measurement kinetics of the functional layer on its gold surface (thiolated PEG and IgG-binding peptide), as well as flow-through performance during capture of HCPs. The QCM biosensor platform was iterated from a single sensor into a multiplexed array with microfluidic channels to enable continuous and rapid detection of HCPs and other target analytes in complex medias. The device behavior was analyzed using computational fluid dynamics of low volume feeds, and comparisons were made between single QCM sensing and a scalable multi-array QCM system.
Wednesday
PAT strategies for modelling and control of single-pass tangential flow ultrafiltration (SPTFF) in continuous manufacturing of monoclonal antibodies
11:10am - 11:30am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 04
Garima Thakur, Presenter, IIT Delhi; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Formulation of biotherapeutics using single pass tangential-flow filtration (SPTFF) is a critical step in continuous manufacturing processes for many drugs including monoclonal antibodies (mAbs). The concentration of the mAb in the final formulation is a critical quality attribute which affects safety and efficacy. Modern SPTFF modules consist of multiple smaller membranes connected in different series and parallel configurations to increase the overall membrane area and residence time in order to achieve high concentration factors in a single pass. The final concentration of the drug is determined by the flux across the module in a single pass, with no scope for repeated recirculation as in typical batch-mode ultrafiltration. Therefore, it is critical to design the membrane configuration and select the operating parameters such that the concentration target is met in a single pass. The present work leverages the gel polarization model of protein ultrafiltration to develop a model for the permeate flux vs. time profile of a single membrane inside an SPTFF module. The single membrane model is then used as a building block to model complex SPTFF configurations and facilitate in-silico design of customized SPTFF configurations resulting in up to 40% savings in membrane area.
We also propose a strategy to control the output concentration of a continuous ultrafiltration step regardless of variations in the volume or concentration of the feed material. Scheduling algorithms for the filtration runs and cleaning cycles are developed, accounting for the expected cycle times of different unit operations in the continuous train. An empirical model of the design space is developed in the range of feed of 1-50 g/L and 10-210 mL/min. The control strategy leverages in-line concentration, flowrate and pressure sensors, including near infrared spectroscopy (NIRS) flow cells to measure the concentration of mAb. The control elements are a permeate pump and a variable control valve. The proposed system is the first complete approach that integrates process understanding, advanced monitoring sensors, and control strategies to consistently achieve concentration targets over long continuous campaigns.

Wednesday
Development of In-line and on-line process analytical technology tools for high concentration biologics processes
11:30am - 11:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Downstream unit process operations used in biological drug manufacturing have changed significantly in recent years. Biopharmaceutical companies must support a large, diverse portfolio, across different manufacturing sites, with many programs requiring high concentration to meet clinical dosing for subcutaneous injection. In particular, the ultrafiltration and diafiltration (UF/DF) process operation, the final step in biologics processing, has presented significant challenges. To ensure process consistency and provide process validation, Process Analytical Technology (PAT) can be a valuable addition to the manufacturing process. Size exclusion chromatography (SEC) based PAT methods were developed for a liquid chromatography system with built-in autosampler. These methods measure the concentrations of the excipients arginine, histidine and high molecular weight species, and were found to be comparable to orthogonal offline methods. An in-line A280 monitoring instrument with variable pathlength extension technology was tested with several portfolio assets, and found to be comparable to offline A280 method up to 260 g/L. These PAT applications are setup as a platform system for monitoring of multiple attributes over a wide dynamic range. In summary, these PAT tools are suitable at present for in process monitoring during GMP and commercial manufacturing, with future potential for real-time feedback control of the process.
Challenges in Developing Novel Modalities: Challenges in Developing Novel Modalities
10:30am - 12:10pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 05
Cesar Calero Rubio, Organizer, Sanofi Genzyme; Mary Krause, Organizer, Bristol Myers Squibb; Krishna Mallela, Organizer, Univ of Colorado Denver; Thomas Tolbert, Presider, Univ of Kansas; Kiran Bangari, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Advances in biotechnology, increased understanding of biological systems and new production techniques have enabled several powerful novel modalities for therapeutics including antibody drug conjugates (ADC), bispecifics, viral or non-viral gene therapy, fusion proteins, CAR-T cell therapies, oligonucleotides, nanobodies and vaccines. This together with the discovery of new biological targets and the rise of public health emergencies like the COVID-19 pandemic has increased the motivation to rapidly develop these powerful new modalities into approved therapeutics to treat high unmet needs in an effective way. This session will focus on the developmental and regulatory challenges of manufacturing novel therapeutic products and accelerated timeline challenges encountered in rapid development of therapeutics including COVID-19 therapeutics. Broad overviews discussing all of the challenges associated with developing a particular modality, or more detailed talks discussing specific challenges encountered during analytical characterization, upstream and downstream processes development, formulation development, validation of new modality processes and regulatory submission of viral, non- viral gene therapy, cell therapy, nanoparticles, fusion proteins, oligonucleotides and antibody drug conjugates, CAR-T cells and vaccines would be appropriate for this session.

Wednesday
Challenges & opportunities for the development of parenteral products of novel modalities
10:30am - 11:10am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
A huge number of modern therapeutic modalities must be administered parenterally in order to achieve systemic action, including biologics, oligonucletotides and cell & gene therapy.
Whilst formulations for antibodies become more established and commoditized, novel formats including bispecifics or fusion proteins may pose different challenges. Viral vector or cell therapy formulations have to a large extend not been exploited at all, which often leads to (deep) frozen storage of such products, leading to significant challenges for distribution and usability alike.
This talk aims to discuss some pharmaceutical challenges - and opportunities - for the development of modern biologics, including therapeutic proteins, gene and cell therapy.

Wednesday
Withdrawn
11:10am - 11:30am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Wednesday
Withdrawn
11:30am - 11:50am USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Wednesday
Design and directed evolution of conformation- and sequence-specific nanobodies against tau amyloid aggregates
11:50am - 12:10pm USA / Canada - Eastern - August 25, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Amyloid aggregation is associated with several devastating neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. Conformational antibodies, nanobodies and other affinity proteins that selectively recognize amyloidogenic aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. However, it remains challenging to generate such antibodies with strict conformational and sequence specificity in a systematic and predictable manner. To address this challenge, we have sought to develop robust approaches for generating nanobodies that selectively recognize tau amyloid fibrils. First, we have developed novel synthetic nanobody libraries that incorporate amino acid diversity into both their frameworks and complementarity-determining regions in order to generate nanobodies with diverse binding properties and epitopes. Second, we have displayed these libraries on the surface of yeast and performed selections using magnetic- and fluorescence-activated cell sorting. We find that this approach leads to strong enrichment for distinct types of nanobodies against tau fibrils. Third, we have affinity matured lead nanobodies using multiple mutational strategies and a novel nanoparticle-based antigen presentation method. We find that these strategies can be used to isolate conformational nanobodies with high affinity and specificity. We expect that the nanobodies and methods developed in this work will accelerate the biomedical targeting of pathological aggregates of tau and other amyloidogenic proteins.