Reset

Advanced Filters
Advances in Chromatographic Separations for mAbs: Advances in Chromatographic Separations for mAbs
10:30am - 12:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Melissa Holstein, Presider; Stefano Menegatti, Presider
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This session hosts contributions focusing on practical and theoretical approaches that advance and optimize antibody purification platforms. Topics include broadening platform applicability across the full range of (i) antibody sources (e.g., mammalian, insect, and yeast cells, plant extracts, etc.); (ii) novel process technologies and modes of chromatographic operation designed to monitor and control antibody-related variants (e.g., charge, glycosylation, etc.); (iii) mechanistic and molecular-level characterization of antibody-related product variants (e.g., aggregates, clipped forms and fragments, etc.); (iv) clearance of impurities (e.g., difficult-to-remove HCPs, viruses and endotoxins, etc.); (v) continuous and flow-through modes of purification for process intensification; (vi) scale-down methodologies to evaluate platform fit for antibody candidates, and (vii) scale-up/process implementation to enable rapid and robust process transfer to manufacturing facilities.

Sunday
Principles of CHO cell culture harvesting using novel synthetic fiber chromatography
10:30am - 10:50am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Mammalian cell culture continues to be the workhorse for expression of high value recombinant therapeutic protein modalities. The first step in the recombinant biotherapeutic process is harvesting cell culture fluid containing the product. Conventional approaches for performing this unit operation utilize a combination of depth filtration, centrifugation, and membrane filtration. These technologies utilize differences in density and in size as the principles of separation. As cell culture processes are intensified to yield higher cell densities and product titers, the ability to effectively harvest the cell culture fluid with the consistency and scalability required becomes challenging.

Here we present a novel single stage harvesting approach that utilizes synthetic fibrous anion exchange (AEX) chromatography to efficiently separate the cells, cell debris, and DNA by charge from the harvested cell culture fluid containing the target biotherapeutic product. Precision quaternary ammonium (Q) functionalized polypropylene fiber bed and a 0.2 μm polyether sulfone membrane in a capsule enables a single stage clarification process of low to high-density cell culture with high recovery, and high fidelity of soluble and insoluble contaminant separation. This new clarification approach enables >95% mAb product recoveries, consistent cell loading capacity, and predictable clarification from discovery to commercial manufacturing scale. Furthermore, open structured fibrous media enables low pressure operation with minimal cell shear.

Sunday
Mechanistic understanding of product binding in protein A chromatography for monoclonal antibodies
10:50am - 11:10am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Recent progress in Protein A chromatography has enabled both high productivity and high resin capacity utilization. However, under high protein loading conditions, we observed significant yield losses of several mAbs during a post-load wash step, which is commonly applied between product load and elution to remove impurities such as host cell proteins (HCPs) and DNA. We have investigated this behavior at three levels. At the column level, we performed breakthrough experiments and chromatography mechanistic modeling to understand protein adsorption and transport properties in various solution conditions. At the resin particle level, we measured adsorption isotherms in high-throughput mode to obtain the protein binding affinity, protein interactions and maximum binding capacity. At the molecular level, we performed isothermal titration calorimetry to gain insights into the protein binding strength by measuring the adsorption enthalpy. Additionally, we have evaluated the effects of various modifiers in the wash step on product yield and impurity clearance. Overall, this work reveals the underlying mechanism of product yield loss observed in various wash conditions and provides practical insights in optimizing these conditions for Protein A chromatography.
Sunday
Influence of excipients on surface-induced aggregation of a monoclonal antibody in cation exchange chromatography
11:10am - 11:30am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
During the development of protein formulations, excipients are tested mostly under accelerated stress conditions. To induce conformational changes and aggregation in a protein formulation and to study the influence of excipients on it, the protein is subjected to e. g. low pH, high temperatures, and different mechanical and chemical stresses in solution. Cation exchange chromatography is generally considered a mild protein purification technique. Recently it has been shown that it can lead to conformational changes and even aggregation of protein when bound to the surface of the stationary phase.
The objective of this project is to establish an analytical screening method for the evaluation of excipients on the conformational stability and surface-induced aggregation of monoclonal antibodies. In contrast to in-solution tests, aggregation is induced by loading the monoclonal antibody on a strong cation exchange column and keep it idle for a specific time before eluting in a salt gradient. Depending on pH, ligand density, hold time, and temperature, the mAb changes into (partially) reversible aggregates. The effects of excipients on this reversible self-association can be evaluated on-column as well as after elution. The results are compared to conventional screening methods, e. g. nano-DSF.
The approach is relevant not only for formulation development but also for solvent-modulated chromatography steps in the downstream processing of proteins.

Sunday
Ion exchange resin ligand density determines mAb stability and recovery
11:30am - 11:50am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Greta Jasulaityte, Presenter; Hans Johansson, Purolite; Daniel Bracewell, UCL Dept Biochemical Engr
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The quality of a purified biopharmaceutical product is judged by its structural and functional characteristics as well as impurity profiles and stability. Product- or process-derived effects on quality attributes can lead to changes in pharmacokinetics and pharmacodynamics of the drug product as well as immunogenicity, often stalling product submission and approval or batch release. Here, the effects of resin ligand density and buffer pH on purity and stability of two mAbs: IgG1 and IgG4, were studied. Monodisperse CEX resin particles with sulfopropyl ligand densities ranging from 0.05 to 0.28 mol/L were manufactured using jetting technology. The purified mAbs were evaluated for changes in their higher order structure including aggregates and isoforms when undergoing binding and elution steps from these resins.
Ligand densities above 0.10 mol/L and pH below 5.0 were a cause of significant peak splitting for the IgG4 molecule, and 60% loss in product recovery. Higher ligand densities provided an increased number of protein-ligand interactions and stronger adsorption. In turn, it led to on-column unfolding and irreversible aggregation detected with a novel fluorescent dye method. Loss of protein structure was confirmed when a stabilizing agent, arginine, was introduced into the process buffers resulting in a significant increase in product recovery. IgG1 was found to be unaffected by the changes in ligand density as it was more thermally and chemically stable than IgG4 resulting in overall higher step yield of >80%.
The effect of ligand density and buffer pH on product recovery and purity were heavily dependent on the general stability of the molecule. Resins with lower ligand densities are recommended for the processes where buffer additives and pH fail to provide desired results.

Sunday
Factors affecting robustness of anion exchange chromatography: Selective retention of minute virus of mice using membrane media
11:50am - 12:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Mobile and stationary phase factors were investigated in order to identify conditions for effective capture of minute virus of mice (MVM), a potential adventitious contaminant in biomanufacturing, using anion exchange membrane chromatography (AEX). The initial study was conducted for Membrane A for a range of feed conditions using bovine serum albumin (BSA) as a model protein mimicking acidic host-cell proteins (HCPs). The effects of pH (~6-8), salt concentration (~0-150 mM NaCl) and level of BSA (~0-10 g/L) were systematically investigated. It was found that higher BSA concentration has the most negative impact on MVM binding followed by the increased conductivity of the feed solution. The effect of pH on MVM binding is also detected but has a less impact compared to other two factors in the range of feed conditions investigated. In addition to Membrane A, three other AEX membranes (Membrane B, C and D) were investigated for MVM binding at a selected feed condition. Based on properties of the membranes investigated, it was found that ligand charge density has the most significant impact on MVM binding performance of AEX membranes from stationary phase perspective.
Sunday
Hitchin’ a ride: Investigation and removal of co-purifying HCPs in monoclonal antibody purification
12:10pm - 12:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Dr. Andrew Robert Wagner, PhD, Presenter, Just - Evotec Biologics; Yuko Ogata; Rosalynn Molden
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Removal of host cell proteins (HCPs) presents a challenge to downstream processes since host organisms can produce thousands of proteins with a range of biophysical characteristics during biologics production. As a result, HCPs that co-purify with the target therapeutic molecule often require specialized approaches for robust clearance. While ELISA assays are commonly used to measure HCP levels in process intermediates, mass spectrometry is becoming increasingly relevant to fundamental understanding by revealing individual HCPs which are co-purifying alongside target molecules. Here we investigated the HCP-molecule interaction by examining the composition of HCPs associated with 7 therapeutic proteins. A unique collection of HCPs was associated with each therapeutic molecule, suggesting the HCP interaction is strongly influenced by both the molecular sequence of the target protein and the associated biophysical or surface properties. While the number of unique HCPs varied widely across the set, there was a common group of 13 HCPs which accounted for majority of the content. Notably, these common HCPs were primarily extracellular proteins or integral membrane proteins, suggesting HCPs from continuously perfused bioreactors are derived from the host organism secretome. This study thus shows the value of building a library of HCPs observed across molecules to ascertain what types of HCPs are associated with certain cell lines, molecular motifs, or process conditions, and which among those factors might present a challenge for HCP removal. The degree of HCP removal via chromatography and filtration unit operations demonstrated the effectiveness of a platform approach despite the diverse HCP profiles within the set. Coupling plate-based resin screens with mass spectrometry analysis illuminated options for removal of high-risk or hard-to-remove HCPs which co-purify with the product. This work demonstrates how technology that identifies and quantifies specific HCP impurities can elegantly guide the fine tuning of downstream processes.
Biomedical Technologies: Imaging, Diagnostics, & Other Integrative Approaches to Study & Model Diseases: Novel approaches to diagnose and study disease
10:30am - 12:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
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.

Sunday
RNA extraction-free rapid detection of SARS-CoV-2 in clinical samples based on lateral flow assay
10:30am - 10:50am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Ketan Dighe, Presenter, University of Maryland Baltimore County; Parikshit Moitra; Maha Alafeef; Dipanjan Pan
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The coronavirus disease 2019 (COVID-19) pandemic has highlighted the major shortcoming of healthcare systems globally in their inability to diagnose the disease rapidly and accurately. At present, the molecular tests (RT-PCR) based approaches to diagnose COVID-19 use reverse transcriptase to create and amplify cDNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Although molecular tests are reported to be specific, false negatives are common. Furthermore, all these tests require a step involving RNA isolation which does not make them POC tests in the true sense.
Here, we report an amplification-free nucleic acid test (NAT), implemented on a lateral flow strip and devoid of a RNA isolation step, which allows rapid diagnosis of positive (nCOV/COVID-19) cases at POC. The assay uses highly specific fluorescein isothiocyanate (FITC) and biotin labeled antisense oligonucleotides (ASOs) as probes that are designed to target N-gene sequence of SARS-CoV-2. Additionally, we used cysteamine capped gold nanoparticles (cyst-AuNPs) for signal augmentation without an additional operation step in lateral flow strip. Without large-stationary equipment and highly trained staffers, the entire contiguous sample-to-answer workflow takes less than 30 minutes from a clinical swab sample to a diagnostic result. Moreover, the assay achieved an accuracy and specificity of almost 100% when tested with 30 clinical samples and confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit.
In summary, we have designed and tested a nucleic acid test implemented in a lateral flow strip for SARS-CoV-2 RNA detection that enables one to interpret the diagnostic results rapidly, accurately, and visually from clinical samples without professional instruments and expertise. We anticipate that our inexpensive amplification-free detection of SARS-CoV-2 RNA could facilitate a platform for rapid and POC diagnosis of COVID-19 and other emerging infectious diseases in resource deficient areas.

Sunday
Tissue engineering is a dichotomy of scaffold-based and scaffold-free cultures. Scaffold-based cultures form highly organotypic structures but with low uniformity and throughput. Conversely, scaffold-free cultures create consistently sized and shaped cell aggregates with limited spheroid-like structure and function, thus restricting their use for accurate disease modeling. We hypothesized that combining aspects of each culturing format, we would produce highly organotypic structures of consistent size and shape for use in pulmonary modeling. To this end, we created a novel easily scalable, minimalistic design for a micropatterned hydrogel dish that increases reliability and efficiency in 3D cell culture. Our suspension cultures are a unique improvement on current methods to produce organotypic structures. By using soluble non-gelling concentration of basement membrane (BM), we created an organotypic lung model from three stable cells representing epithelial, vascular, and stromal cell populations in substantially less time a currently reported methods with similar properties. We observed that soluble BM promotes emergence of lumina comparable to mammalian lung airspaces. Using hypoxia induction techniques, we provide evidence for formation of branching, perfusable vasculature in pulmonary aggregates supplemented with soluble BM. Aside from these structural traits, we observed increased proliferation, survival, and 3D growth of aggregates. These results were established using IF, biotinylated-Matrigel and proteomic techniques. As proof of concept, we applied this method in modeling of lung fibrosis using TGF-Beta induction followed by testing two investigational antifibrotic drugs. Our results demonstrate a novel 3D culture method that creates organotypic models from stable cell lines. We anticipate this technology to pioneer creation of novel suspension-based organoids fostering consistent, expedited 3D culture. Recently we used our cells in a customizable miniaturized high-resolution 3D printed microfluidic device capable of 2-fold serial dilutions which we validated using a permeabilization assay. Proving these technologies have the potential to expedite patient-derived organoids for personalized drug screening using lung-on-a-chip assays.
Sunday
Effects of fluid shear stress on reactive oxygen species generation, stemness, and epithelial-to-mesenchymal transition
11:10am - 11:30am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Spenser Brown, Presenter; Juliana Bates; Alexandra Avera; Yonghyun Kim, The University of Alabama
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Most cancer deaths are caused by secondary metastasized tumors initiated by circulating tumor cells (CTCs). These cells are in a dynamic microenvironment where fluid shear stress (FSS) is a prominent force with myriad of consequences, including reactive oxygen species (ROS) generation. It is believed that there exists a correlation between CTCs and cancer stem cells (CSCs) and that this resemblance supports CTCs to survive the metastatic process and proliferate afterwards for colony formation to form a secondary tumor. One stem cell-like quality is the ability to undergo the epithelial-to-mesenchymal transition (EMT). In this study, triple-negative subtype breast cancer MDA-MB-231 and luminal subtype MCF7 cells were subjected to a prolonged FSS in a spinner flask as an in vitro model of CTCs. Post-FSS, the cells were analyzed via flow cytometry and qRT-PCR for ROS generation, CSC, EMT, and Hippo markers. Though MDA-MB-231 did not show a dynamic CSC shift, there were significant changes in ROS, EMT, and Hippo markers (p<0.05). Additionally, MCF7 also showed significant changes in ROS and EMT markers. Notably, both cell lines exhibited the mesenchymal-to-epithelial transition (MET) signature upon recovering from exposure to FSS. These findings indicate that the baseline stemness quality (or lack thereof) and phenotype of each cell line affects how they respond to induced biophysical forces. Furthermore, this shows a possible relationship between mechanotransduction, the Hippo pathway, and the induction of EMT in breast cancer cells.
Sunday
Biofilm-functionalized single-walled carbon nanotube probes for high throughput screening of enzymes that degrade Pseudomonas aeruginosa biofilms
11:30am - 11:50am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogenic bacterium known for causing nosocomial infections in immunocompromised individuals. The ability of P. aeruginosa to develop biofilms contributes to its resistance to antimicrobial agents and immune defenses, making the treatment of infections more difficult. Recent strategies to combat biofilm infections include enzymatic treatments to degrade the biofilm extracellular polysaccharide matrix (EPS), allowing for better diffusion of antibiotics. While it is crucial to understand biofilm degradation, current methods for quantitatively measuring enzymatic degradation of the extracellular matrix are time-consuming and not amendable to high-throughput screening of suitable enzyme candidates. Herein, we present biofilm-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on biofilm EPS. The biofilms are harvested and used to suspend the hydrophobic SWCNT via tip sonication. The extent of biofilm EPS degradation is quantified by monitoring quenching of the SWCNT fluorescence caused by changes to the SWCNT local dielectric upon enzymatic degradation. We used this platform to screen thirteen hydrolases with varying bond breaking selectivity against a panel of wild type P. aeruginosa and various of its mutants deficient in one or more EPS polymer or altered in the amount or type EPS produced. Our results indicate that the enzymes with selectivity towards α-(1,4), β-(1,4), and β-(1,3) glycosidic linkages, such as lyticase and amyloglucosidase, can most effectively disrupt the EPS. Next, we performed concentration-dependent studies of enzymes on two of the most common strains found in Cystic Fibrosis (CF) environments (PDO300-alginate overproducer and MJK8-small colony variant) and extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model. We found a good model fit (R2 ranging from 0.71 to 0.97) of the dynamic data from six enzymes tested at five different concentrations each using the same five parameter model. Reaction rates (turnover rate) are dependent on enzyme concentration and ranges from 2.16e-9 to 2.80e-3 *s-1 per mg/ml of enzyme. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of the hydrolase panel. This platform should enable faster discovery and design of enzyme-based therapies.
Sunday
Design and use of tissue-like neural probes to study the progression of pathological hallmarks of Alzheimer's disease
11:50am - 12:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Theodore Zwang, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In this work we study pathological hallmarks of Alzheimer’s disease by taking advantage of tissue-like neural probes, mesh electronics, that are designed to reflect the brain environment in which they are employed. Mesh electronics are designed to minimize structural, mechanical, and topological differences with brain tissue, which allows for the electrophysiological study of the same individual neurons and their circuits over many months. These data help understand how aging with the presence of different pathological hallmarks of Alzheimer's disease relate to the progression of neuronal and circuit-level dysfunction in rodents, and may be extended to understand the how other biochemical processes can influence neurophysiological changes over long periods of time in vivo.
Sunday
A noninvasive multiplexed biomarker-based bladder cancer test to improve patient follow-up frequency and compliance
12:10pm - 12:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Bladder cancer (BC) is a deadly and highly recurrent disease that requires extensive routine follow-up testing to ensure progression is caught early. The gold standard techniques for disease diagnosis and monitoring are cystoscopy and urine cytology cause patient discomfort and the invasive nature of cystoscopy, there is decreased patient compliance with frequency of exams, leading to delayed detection, classification, and treatment. There is an urgent unmet need for a non-subjective BC follow-up test that can distinguish differences in cell function (healthy vs. oncogenic) even when cellular appearances are the same (early-stage and stage-specific detection). Early Is Good, Inc., is taking a biomarker-based approach for (a) initial BC diagnosis, including non-visible tumors missed by cystoscopy, (b) monitoring for recurrence/progression, (c) reducing the frequency of expensive and uncomfortable cystoscopies, and (d) risk stratification and informing patient-specific treatment plans. Early Is Good has developed a novel localized surface plasmon resonance enhanced biolumicensence based biomarker detection platform with unprecedented femtomolar sensitivity. we are leveraging this platform to create “BCDx,” a highly sensitive, highly specific point-of-care follow-up BC test. BCDx simultaneously quantifies a panel of five different biomarkers from patient urine, including two proteins (NMP-22, BTA), two microRNAs (miR-10b, miR-145), and one long non-coding RNA (UCA1) in order to diagnose BC and BC stage in as little as 15 minutes. We observed femtomolar LOD (s.d. 3.4–4.9 fM) with artificially spiked urine samples. To confirm that the presence of some biomarkers, especially those expected to be elevated in BC samples, does not interfere with quantification of others, we measured all five biomarkers simultaneously in a multiplexed assay. To demonstrate the efficiency as an at home diagnostic tool, we analyzed the urine of 45-bladder cancer patients without any sample processing steps and 45 normal control patients. Importantly, our noninvasive biosensing approach is capable of quantifying statistical differences using 5 biomarker levels between BC patient urine samples (Stage T1-T4) and healthy samples. Early Is Good will be ready to apply BCDx testing to a larger patient population to further validate BCDx as an effective, noninvasive, and robust follow-up and diagnostic test for the BC community.
Advances in Protein Engineering 1: Advances in Protein Engineering
10:30am - 12:35pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Nik Nair, Organizer; Zhe Rui, Organizer, UC Berkeley; Dr. Julie Champion, Presider, Georgia Institute of Technology; Xuejun Zhu, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

As protein engineering becomes more widely adopted for different applications, ranging from therapeutics to energy to materials, new technologies are needed to enable faster and more efficient engineering of proteins. This session will focus on advances in protein engineering technologies that are currently being developed in academia and industry. Relevant topics include, but are not limited to, new platforms for accelerating protein engineering, innovations in library generation, new screening approaches and high throughput technologies, and advances in computational strategies for protein design. Abstracts that discuss new tools for introducing non-standard amino acids or post-translational modifications and new approaches for engineering protein stability, functionality, and manufacturability are also welcome.

Sunday
Introductory Remarks
10:30am - 10:35am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Sunday
Bio-orthogonal non-canonical amino acid tagging to understand bacterial persistence
10:35am - 10:55am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Phenotypic heterogeneity in populations of isogenic bacterial cells includes variations in metabolic rates and responses to antibiotic treatment. In particular, sub-populations of “persister” cells exhibit increased antibiotic tolerance. Understanding the mechanisms that underlie bacterial persistence would constitute an important step toward preventing and treating chronic infections. In this work, we demonstrate the use of Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) for time- and cell state-selective proteomic analysis of cultures of Pseudomonas aeruginosa that exhibit persistence upon antibiotic treatment. Two methods were developed in the P. aeruginosa strain PA14 by treating cells either with L-azidohomoalanine (AHA) or with L-azidonorleucine (ANL). Comparing the proteomic profiles of untreated cells and persister cells showed that proteins involved in the biosynthesis of pyochelin, a secondary siderophore involved in bacterial iron acquisition, were down-regulated in the dormancy phase and up-regulated in the regrowth phase. Furthermore, regrowth behavior is altered by a gene knockout in the pyochelin pathway, but not by a knockout in the biosynthesis of pyoverdine, another siderophore generally thought to be predominant in bacterial iron acquisition. We are exploring the implications of these results with respect to the mechanisms of persistence-related phenotypic state switching.
Sunday
Solvent-free liquid proteins: Enhancing proteins through surface engineering
10:55am - 11:15am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Dr Alex Brogan, Presenter, King's College London
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Surface engineering of proteins, to yield protein-polymer surfactant nanoconjugates, has been demonstrated as a robust method for synthesizing protein-rich biofluids that are devoid of any solvent. This new class of biomaterial has been shown to be a promising new technology where enzymes have been stabilized in non-aqueous environments.

Using a variety of spectroscopic and scattering techniques, these novel biomaterials have been shown to allow for extreme enzyme thermal stability, stability against aggregation, retained dynamics, and enhanced function. We later showed that these protein-polymer surfactant nanoconjugates are soluble in both hydrophilic and hydrophobic ionic liquids, and we demonstrated that biomolecule architecture can be preserved in the non-aqueous environment. Furthermore, the solubilized protein displayed improved thermal stability as compared to aqueous solutions. Through surface modification of the enzyme glucosidase we were able to demonstrate that once solubilized in ionic liquids, the enzyme has significantly improved activity at 120 °C, and otherwise unseen activity towards water insoluble cellulose. Importantly, this demonstrated that it was possible to reduce the number of enzymes require for cellulose degradation from 3 to 1.

Here, I will show recent work involving avidin and the M13 bacteriophage. Using avidin as a proxy for therapeutic proteins, we demonstrate that solvent-free biofluids can maintain the structure of this protein and preserve its biotin binding ability at significantly elevated temperatures; the denaturation temperature of solvent-free avidin was measured as 139 °C. As a result, accelerated aging studies showed no changes in structure would be expected for storage up to 160 days at room temperature. Using the bacteriophage M13, we also show that this methodology can be extended, in conjunction with ionic liquids, to create thermally robust ionogels with high-aspect ratio scaffolds.

Solvent-free liquids of avidin and the M13 bacteriophage demonstrate the increasing versatility of creating soft biomaterials via surface engineering of proteins. These recent results indicate that this biomaterial could be used as a platform for removing the requirement for the cold chain for protein-based therapeutics, as well as providing routes to introduce genetically engineerable biocompatible scaffolds for non-aqueous soft materials.

Sunday
Design of high affinity, specificity, and stability pro-apoptotic stapled peptides using bacterial cell surface display
11:15am - 11:35am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
There is currently a wealth of disease-associated proteins that are “undruggable”: inside the cell and inaccessible to large biologics but lacking small molecule binding sites. Stapled peptide therapeutics, short chains of amino acids where two are covalently crosslinked, are an emerging therapeutic framework that fill this gap. However, their discovery is reliant on solid-phase synthesis, limiting throughput and evaluation.
We employ a novel technique known as Stabilized Peptide Engineering by E. coli Display (SPEED) to rapidly characterize ~109 stapled peptide therapeutics. In this technique, libraries of DNA encoding peptides are designed by mining linear peptide data. Stapled peptides are formed directly on the cell surface by incorporation of azide residues in place of methionine and reaction via copper catalyzed click chemistry. Finally, cells are sorted for given molecular properties by incubating with fluorescently labeled target protein.
In this work, we demonstrate SPEED’s utility by applying it to the Bcl-2 protein family, important regulators of cell death. First, we utilized on-cell staple scanning to identify staple locations that maintain target binding. Interestingly, some of these locations resulted in changes in specificity for different Bcl-2 proteins. Next, incorporation of Next Generation Sequencing enables quantitative measurement of affinity and specificity, streamlining downstream analysis. Finally, peptide stability was optimized by treatment with peptide-degrading enzymes before fluorescent antigen treatment.
To our knowledge, this work represents the first high-throughput study of stapled peptide Bcl-2 inhibitors. Peptides with these improved drug-like properties may promise better selective treatment for cancer with in vitro and in vivo validation of efficacy. This approach provides a framework to discover potent stapled peptide inhibitors towards other important protein targets.

Sunday
AUBIE: Overview of an algorithm for the rapid de novo design of binding proteins
11:35am - 11:55am USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Proteins are polymers built from the small, monomeric units of amino acids. These large biomolecules are the fundamental building blocks of all living organisms. They perform a wide range of functions in organisms such as DNA replication, providing cellular structures, catalyzing biochemical reactions, assisting in cell signaling, and immune responses. In the last several decades, there has been a steady improvement in our ability to computationally predict and design their structures. In the last year, the success of Alpha-Fold 2 demonstrated that the rules of protein structures are now sufficiently well understood that they can be predicted with an accuracy matching experimental methods. Now, the focus of computational protein design and engineering is shifting to better model protein interactions and binding.
The archetype of binding proteins are antibodies (Abs), also known as immunoglobulins (Igs). These immune system proteins are responsible for protecting the body against infectious, disease causing organisms. They are also widely used as therapeutics, with an annual global market well in excess of $100 billion. Even though Igs are critical for treating diseases, the conventional approach of designing them in the laboratory can be expensive, time consuming, and remains a matter of luck. An alternative to this experimental approach is to use computational methods to design antibodies and other proteins with similar binding mechanisms (e.g. nanobodies, 10th Type III Fibronectin (10Fn3) domains, etc.).
The Pantazes research group developed an Algorithm for Ultra-rapid Binding Interaction Engineering (AUBIE) for the fast, de novo design of therapeutic binding proteins. The key focus of the algorithm is on the optimal placement of hotspot binding interactions, enabling the design of high-affinity protein binders. This presentation will describe the algorithm’s database generation and design protocols, as well as discuss the features of designed proteins against a series of peptide and protein antigens, including the receptor binding domain of SARS-CoV-2. The presentation will finish with a vision for how these approaches form the basis for the rapid, rational design of functional proteins.

Sunday
Engineering and design of a minimal metalloproteinase inhibitor scaffold
11:55am - 12:15pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Linh Do; imam sanousi; Mari Rita Toumaian, University of Nevada, Reno; Alexander Bolt; Maryam Raeeszadeh Sarmazdeh, Presenter, University of Nevada, Reno
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of metalloproteinases (MPs), which are zinc-dependent endopeptidases with key roles in degradation and remodeling of the extracellular matrix (ECM). Dysregulation of MPs is long known for its contribution to several diseases such as cancer, neurological disorders, and cardiovascular diseases. TIMPs, a protein family of four members in humans, show a spectrum of binding affinity and selectivity to different MPs as well as other unique cell signaling binding partners, despite TIMPs having high sequence and structure similarity. TIMPs are comprised of two domains, with the N-terminal domain known as the main inhibitory domain. Understanding the underlying binding mechanism of TIMP-MP binding based on sequence and structure has a great importance in developing the next generation of effective protein-based therapeutics targeting MPs.

We used yeast surface display and protein engineering techniques such as DNA shuffling and directed evolution to identify a minimal inhibitory domain and fragment in the TIMP family. First, we displayed full-length and N-terminal domains of the TIMP family (TIMP-1, -2, -3, -4) on the yeast surface and analyzed binding and expression using flow cytometry. Various TIMPs displayed varied levels of expression and MP binding on the yeast surface, with full-length TIMP-3 having the lowest binding and expression. Additionally, we screened a scrambled TIMP library, made using DNA shuffling within the TIMP family, for expression and MP binding via fluorescent-activated cell sorting (FACS). Interestingly, several scrambled TIMP fragments with sizes as low as 40 amino acids were isolated after rounds of FACS screening, with maintained or improved binding to MMP-3 catalytic domain. The study allowed us to explore any TIMP motifs involved in binding to MP or other cell signaling targets, and hypothetically engineer more functional, tissue-penetrable and selective peptide drug candidates.

Sunday
Profiling the substrate proteins of RING-type E3 ubiquitin ligase by the engineering of orthogonal ubiquitin transfer (OUT) cascades
12:15pm - 12:35pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Li Zhou, Presenter; In Ho Geong; Savannah Jacobs; Sazid Hassan; Dr. Jun Yin, Georgia State University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) and RING finger protein 38 (RNF38) are members of the RING family of E3 ubiquitin (UB) ligases and regulate key processes in the cell. Through the E1-E2-E3 cascades, Cbl-b and RNF38 can transfer UB to cellular proteins and regulate their stability, subcellular localization, and interaction with other proteins. Identifying the ubiquitination targets of Cbl-b and RNF38 holds the key to decipher their roles in cell regulation. In this study, we used phage display to engineer the Ring domains of the two E3s for their incorporation into an orthogonal UB transfer (OUT) cascade to identify their substrate proteins in the cell. The OUT cascades consisting of engineered E1, E2 and E3 enzymes enable the exclusive transfer of an engineered UB (xUB) through a designated E3 to its substrate proteins. By affinity purifying xUB-conjugated proteins from cells expressing the OUT cascade of an E3 and identifying them by proteomics, we would be able to map the UB transfer pathways from the E3 to its cellular targets. So far, we have successfully constructed the OUT cascade of RNF38 and used it to profile its substrates in HEK293 cells. The newly acquired substrate profile of RNF38 enables us to discover potential roles of the E3 in cell cycle regulation and other cellular processes. We also expect our engineering of the RING domain of Cbl-b will generate an OUT cascade of the E3 for elucidating its functions in the cell. Overall, we have demonstrated that the OUT cascades we generated with RING E3s are powerful tools for profiling E3 substrates and mapping the cellular circuits mediated by the E3 enzymes.
Sunday
BIOT Young Investigator Award
01:20pm - 02:00pm USA / Canada - Eastern - August 22, 2021
Division: [BIOT] Division of Biochemical Technology
Session Type: Networking Events - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This year's winner of the BIOT Young Investigator Award is Mark Blenner. The talk is entitled: Domesticating Oleaginous Yeast for Biochemical Production from Low-Value and Waste Substrates Dr. Mark Blenner is an Associate Professor of Chemical and Biomolecular Engineering at the University of Delaware. Prior to that, he was the McQueen Quattlebaum Associate Professor of Chemical and Biomolecular Engineering at Clemson University. He received his PhD in Chemical Engineering from Columbia University in 2009 and completed three years of postdoctoral training as an American Heart Association Postdoctoral Fellow and an NIH NRSA Postdoctoral Fellow at Harvard Medical School and Children’s Hospital Boston. In addition to winning the 2021 ACS BIOT Young Investigator Award, Dr. Blenner has won the Presidential Early Career Award for Scientists and Engineers (PECASE), the 2020 South Carolina Governor’s Young Scientist Award for Excellence in Scientific Research, the 2019 Clemson University Junior Researcher of the Year, and several Young Faculty Awards from DARPA, NIH, NASA and the Air Force. His research is broadly focused on engineering biomolecular and cellular systems for the production of fuels, chemicals, enzymes, biopharmaceuticals and biosensors. Recently his group has started to focus using multi-omics methods to improve engineered cell lines for the biomanufacturing setting.

Downstream Processing: Advances in Chromatographic Separations for Novel Antibody Structures & Drug Conjugates: Advances in Chromatographic Separations beyond mAbs 1
02:00pm - 04:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Asif Ladiwala, Presider, ‍ ; Dr. Brandon Coyle, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This session calls for papers focused on the downstream processing of novel antibody structures which may include, but are not limited to, bispecific antibodies, antibody drug conjugates (ADC), single-chain variable fragments (scFv), antigen binding fragments (Fab), novel antibody structures, or other protein conjugates. The scope may range from theory/modeling, early stage screening, early/late stage development, process scale-up, and/or large-scale manufacturing. The following topics are particularly encouraged and may include HTPD, process optimization, troubleshooting, and/or case studies focused on antibody derivatives or other protein conjugates: 1. Investigations for new drug modalities and novel chromatographic ligands (e.g., affinity, HIC, multimodal), 2. Optimization of conjugation chemistry/unit operations, 3. Purification of conjugation products addressing challenges in removing undesired conjugation byproducts and/or difficult-to-remove impurities, and 4. Creative approaches to handling unstable products.

Sunday
Determinants of capacity of Fc-based antibody structures in protein A chromatography
02:00pm - 02:20pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Soumitra S Bhoyar, Presenter, University of Delaware; Xuankuo Xu; Steven Traylor; Dr. Jing Guo, Bristol Myers Squibb; Sanchayita Ghose; Abraham Lenhoff
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Despite its ubiquitous use, the mechanistic details of PrA chromatography have not yet been characterized fully, and some factors may help in achieving an optimal balance between yield and purity. The investigation here was prompted by a significant reduction in mAb yield during a high-pH wash to improve clearance of host-cell proteins, which raises questions about similar behavior that may arise for other Fc-based structures, such as Fc fusion proteins. The loss of yield was found to result from an appreciable dependence of both the static and dynamic capacities on pH, including a significant decrease at high pH. We investigated further by seeking to distinguish the effects of pH on mAb affinity to the PrA ligand from effects that altered the capacity directly. We prepared Fc fragments by papain digestion and measured the dependence of their static capacity on pH. Similarly to the intact mAb, the capacity of the Fc fragment also drops at high pH, indicating that the high pH yield loss is a property intrinsic to the Fc fragment, with little contribution from the Fab fragments. Next, we fitted the general rate model to the breakthrough and elution data generated using the intact mAb and the Fc fragments to identify and distinguish between changes in mAb-PrA (or Fc-PrA) interactions and mAb-mAb (or Fc-Fc) interactions. The model parameters for the intact mAb and its Fc fragment will be compared, and possible mechanistic interpretations will be discussed.
Sunday
Evaluation of mild pH elution protein A resins for purification of antibodies and Fc-fusion proteins
02:20pm - 02:40pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Felicia Sadikin, Presenter, Biogen; Yamin Fan, Biogen; Wai Keen Chung
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. The use of low pH (3.0-4.0) to elute the molecule and achieve acceptable yield (>85%) can lead to product degradation (e.g. fragmentation, aggregation) for low pH-sensitive molecules. Elution of the product into a neutral pH buffer solution is a valid alternative but results in lower manufacturing flexibility.
In this study, two protein A resins that elute at a mild pH were evaluated and compared against two conventional protein A resins using five mAbs (four glycosylated, one aglycosylated) and an Fc-fusion protein that formed aggregate at low pH. All mAbs showed higher elution pH on the novel resins compared to conventional resins. One novel resin is a universal mild pH elution resin, with elution pH at or above 4.6 for glycosylated mAbs and Fc-fusion protein, with glycosylation contributing to binding affinity. The other novel resin elutes at a pH above those of the commercially available protein A resins for all molecules evaluated, and the elution pH of the antibodies is dependent on their VH sequences. Additionally, the Fc-fusion protein which did not possess Fab domains showed an even larger difference in elution pH between novel and conventional resins, indicating that interaction between the Fab and protein A ligand contributes to retention.
Both novel resins had similar dynamic binding capacities to next-generation protein A resins for all molecules evaluated. Purification runs with cell culture material were performed on all resins with one mAb and the Fc-fusion protein, using optimized elution pH and wash conditions. Compared to the conventional resins, the novel resins had improved HCP and aggregate removal, and a milder native eluate pool pH for both molecules. For the low pH sensitive Fc-fusion protein, product eluate from the conventional resins had higher starting aggregate levels which also increased significantly with time. In contrast, product eluate from the novel resins had lower starting levels of aggregate which remained constant with time, demonstrating that the novel mild elution pH protein A resins provide a good alternative to conventional resins for low pH-sensitive molecules.

Sunday
Protein A resin engineered to allow quantitative elution of human IgG at pH 5
02:40pm - 03:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Hans Johansson, Presenter, Purolite
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Protein A resins designed for large scale purification of monoclonal antibodies were launched more than thirty years ago. The first resins were based on the wild type of Protein A expressed by a pathogenic Staphylococcus aureus strain. Over the 30 years the manufacturing of monoclonal antibodies has grown tremendously and are now the most important group of molecules in the pharma industry. Along with this development, Protein A resins have been significantly improved both with respect to capacity, productivity, and alkaline stability, resulting in a dramatic enhancement of process performance. Current trends in antibody-based therapeutics include the development of a variety of different atypical IgG constructs that in many cases are prone to aggregation, are expressed at very high titer, and contain several product variants. There is thus an increasing interest in resins capable of resolving product variants at the Protein A step and eluting IgG at milder pH conditions compared to current Protein A resins on the market.
This paper will present data from a new Protein A resin that allows elution of IgG, including IgG of the VH3 family at significantly higher pH compared to regular Protein A resins.

Sunday
Withdrawn
03:00pm - 03:20pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Sunday
Coupling high throughout and in silico tools for next generation downstream bioprocess development
03:20pm - 03:40pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Improvements in efficiency in process development can lead to reduced development timelines as well as better process understanding which helps to minimize manufacturing and patient risks. In the last decade, high throughput chromatography screening techniques using plate based or miniature column techniques have moved from proof-of-concept lab studies to commonly practiced techniques in industrial workflows. Concurrently, in silico modeling tools such as mechanistic chromatography modeling and biophysical structure modeling have migrated from predominately research concepts to methods that can aid industrial process development. This talk will highlight ways that the blending of these in silico tools together with high throughput experimentation can lead to more advanced process understanding and next generation development workflows. Specific examples include: i) predicting scale-up chromatography performance from miniature column data with the assistance of mechanistic models, ii) using plate based chromatography screening data to fit mechanistic isotherm models for predictive scale-up, iii) using biophysical homology models to predict chromatography behavior from primary amino acid sequence alone, and iv) beginning to use structure model descriptors to help predict mechanistic model isotherm parameters.
Sunday
Investigating the impact of a novel single stage chromatographic clarification solution on process economics
03:40pm - 04:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 03
Dr Hani El-Sabbahy, Presenter, 3M
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Process intensification continues to be an important trend in the biopharmaceutical industry. The need to respond rapidly to global health emergencies, together with diversification of pipelines mean that the drive for more flexible intensified processes is likely to increase. Development in upstream processes have lead to higher cell densities and impurity levels as process developers strive for higher titers in the bioreactor to increase productivity. This is putting increasing pressure on the downstream process and harvest clarification step to clear these soluble and insoluble impurities.

In this talk we will explore the impact of a novel, single stage, chromatographic clarification solution, which has been designed for high cell density cell cultures, on process economics. The process modelling, conducted using Biosolve, investigates the impact of replacing existing clarification technologies such as depth filtration and centrifugation with this novel technology. Specifically, the impact on overall cost of goods, sustainability and process productivity will be explored.