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Protein Developability; Physical & Chemical Stability : Protein Developability, Physical and Chemical Stability
04:30pm - 06:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Cesar Calero Rubio, Organizer, Sanofi Genzyme; Mary Krause, Organizer, Bristol Myers Squibb; Krishna Mallela, Organizer, Univ of Colorado Denver; Yongku Cho, Presider, ‍ ; Michael Marlow, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

The need for efficacious biotherapeutics under rapidly accelerated timelines has never been more clearly evident. Confident selection of candidates to progress into Development as early as possible is vital to increasing our speed to the clinic and benefiting patients. This session will focus on recent advances across academia and industry in computational and empirical approaches for 1) predicting and assessing therapeutic protein developability and 2) understanding, engineering, and mitigating mechanisms of physical and chemical degradation, colloidal stability, and non-specific interactions. Suggested topics include: strategies for cross-functional candidate selection, including managing the Research/Development interface; use and confirmation of computational (AI/ML), homology modelling, or small-scale predictive assays; design, implementation, and interpretation of high-throughput screening; predicting process fit and impact of process impurities; integrating physical and chemical forced degradation, accelerated and stress assessments, formulation screening, immunogenicity and PK/PD modelling into candidate engineering/selection.

Tuesday
Photo-degradation of therapeutic antibodies: Manufacturing aspects
04:30pm - 04:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
We previously showed that formulated therapeutic antibody drug substance (DS) are sensitive to ultra-violet (UV) light, as well as to visible light (380-700 nm). Exposure to indoor white light from daylight fluorescence lamps during manufacturing process can cause deterioration of product quality, including formation of high molecular weight aggregates, alteration of charge variant profiles, oxidization of tryptophan and methionine residues and darkening DS color/appearance. Here we present our new studies of potential impact factors on DS light sensitivity, such as color light used in DS manufacturing and storage area, DS protein concentration, metal chelating agents in formulation buffers and DS filling amount in disposable bioprocessing containers. These findings will help to assess and mitigate risks associated with light exposure during manufacturing.
Tuesday
Molecular determinants of strong trade-offs between antibody formulation stability and off-target binding
04:50pm - 05:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Successful development of therapeutic antibodies requires high antibody stability, low off-target binding, low toxicity, low viscosity, high solubility and low levels of aggregation. Several of these properties are strongly influenced by the sequence and structural composition of antibody variable regions and especially their complementarity determining regions (CDRs). We are investigating the molecular determinants of various antibody developability properties by systematically mutating the CDRs of a panel of clinical-stage antibodies to alter their charge, polarity and hydrophobicity. We find a surprisingly strong trade-off between antibody non-specific interactions in physiological conditions and antibody self-interactions in formulation conditions. Interestingly, antibodies with strong repulsive self-interactions in formulation conditions (important for low viscosity) typically display high levels of non-specific interactions in physiological conditions (detrimental to bioavailability), and vice versa for antibodies with low levels of non-specific interactions. We find that this behavior can be largely explained by the isoelectric point of the Fv region, as highly positively charged antibodies promote repulsive interactions that promote high antibody stability at formulation conditions (pH 5-6) but strong non-specific interactions with negatively charged biomolecules at physiological pH (pH 7.4), and vice versa for antibodies with negatively charged Fv regions. Finally, we have identified intermediate ranges of Fv isoelectric point that best balance the competing demands of high antibody stability in formulation conditions and low non-specific binding in physiological conditions. We expect that these findings will improve the design and engineering of antibodies and antibody libraries with drug-like properties.
Tuesday
Structural characterization of folding of GbX with focus on intrinsic parameters influence protein stability
05:10pm - 05:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Globins are small globular metalloproteins that contain heme prosthetic groups and have a wide range of biological functions. Recent advancements in sequencing technology allowed for the recognition of novel six-coordinated vertebrate globins. Although these proteins share high structural similarities with pentacoordinate proteins, the presence of the hexacoordination, as well as an intracellular disulfide bridge, make hexacoordinate globins an excellent model for understanding structural determinants that influence protein stability. Previous studies in our group revealed an increased pH stability of hexacoordinate GbX. To provide a further understanding of structural features that contribute to the increased stability of this protein, we combine “in solution” and “in gas” approaches to characterize unfolding mechanism of GbXWT, GbXC65A, and GbXH90V. GbXC65A has similar stability as the wild-type protein towards unfolding by GuHCl with ΔG = 13 kcal mol-1 and 15.4 kcal mol-1 respectively. However, the stability of GbXH90V is significantly reduced, ΔG = 4.8 kcal mol-1. Emission data show that unfolding of GbXC65A is more complex as an unfolding intermediate has been detected at [GuHCl] = 5.0-5.5 M. The TIMS-MS technique also confirmed the high stability of GbX and GbXC65A. GbX is significantly more stable than pentacoordinate myoglobin, (ΔG = 5.0 kcal mol-1) and hexacoordinate Ngb, (ΔG = 7.3 kcal mol-1), suggesting that other factors than the presence of coordination bond between distal histidine and heme iron contribute to the stability of this protein. The comparison of the amino acid sequence of Mb, Ngb, Cygb, and GbX reveals a smaller percentage of nonpolar amino acid residues and an increased amount of charged amino acid residues in GbX. A higher ratio of charged amino acid residues may result in unusual GbX stability which was previously observed in the study of hydrogen bond’s contribution from the side chain residues to protein stability.
Tuesday
An in silico tool for accessing developability : QSAR models for predicting antibody solubilities
05:30pm - 05:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Xuan Han, Presenter, Rensselaer Polytechnic Institute; Qing Chai; Steven Cramer, Rensselaer Polytechnic Inst
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Monoclonal antibodies(mAbs) can often suffer from poor solubilities especially when high concentration formulation is needed for subcutaneous injection. In the early development stage, candidate screening based on experimental characterizations are both material and labor intensive. To facilitate the process, we developed an in silico predictive model using amino acid sequence as input to help researchers determine solubility level of molecules prior to experiments. A pool of antibodies was employed for training the model and solubility data was acquired using PEG precipitation by a high-throughput screening technique (HTS). Refined homology models of these mAbs were then built by taking protein dynamics into account. Molecular descriptors that depict surface properties of antibodies were calculated for each mAb according to their sequences as well as 3D structures. Utilizing these descriptors as input, regression and classification models were developed to help distinguish mAbs that showed desirable solubility from those with poor behavior. Further, we also evaluated our model using external published dataset for further validation. This work not only provides a strategy for antibody early screening based on solubility, but also sheds light into the relationship between protein surface properties and solubility where complicated protein-protein interactions are involved, providing potential guidance for designing mAbs with better manufacturability properties.
Tuesday
Coarse-grained models for rational design of monoclonal antibodies via control of electrostatic protein-proteiniInteractions
05:50pm - 06:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
There is a long standing need for computational tools to predicting protein-protein interactions that can be used to mitigate physical stability of therapeutic proteins. The present work focused on a recently developed coarse-grained molecular simulation approach to predict the experimental second osmotic virial coefficient (B22) for monoclonal antibody solutions as a function of pH, ionic strength, and the protein sequence. Static and dynamic light scattering (SLS and DLS) experiments illustrated very strong electrostatic attractions for a given wild-type (WT) monoclonal antibody in low and intermediate ionic strength conditions. The course-grained model (1bC/D, for one-bead-per-charged-site-and-per-domain) along with a biased sampling Monte-Carlo (MC) simulation was used to identify the most influential charge residues governing electrostatic interactions. This was used to identify all possible single charge-swap and charge-to-neutral mutations potentially impacting self-association of the WT MAb. Twelve variants were made and tested experimentally at six positions by charge-swap and charge-to-neutral substitutions based on the ranking of the B22 scores and their potential impact on binding affinity. SLS and DLS experiments were used to characterize the behaviors of variants under low and intermediate ionic strength conditions. Differential scanning calorimetry (DSC) as well as circular dichroism (CD) were used to assure minimal impacts on conformational stability and secondary structure of variants. Overall, the 1bC/D model simulations provided quantitative or semi-quantitative agreement with the experimental results, and identified multiple variants that were dramatically more physically stable than WT.
Tuesday
Influence of competing pathways on non-enzymatic protein deamidation at a neutral pH
06:10pm - 06:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Protein degradation studies are of interest due to the high demand for protein-based therapeutics in the pharmaceutical industry. However, formulation efficiency is limited by the degradation of the desired products during multiple points of the biomanufacturing process. While the use of additives has significantly reduced degradation, screening of additives and stabilizers is greatly limited by current understanding of the degradation mechanisms. For this purpose, computational models have been developed to quantify the properties associated with each degradation pathway. One such form of degradation is deamidation or the spontaneous conversion of net neutral Asparagine and Glutamine into their acidic counterparts. In this study, we analyzed how selectivity towards specific deamidation pathways was influenced by the interactions between functional groups in model dipeptides using quantum chemistry, statistical thermodynamics, and reaction pathway analysis. Our studies have established trends in thermodynamic and kinetic parameters as a function of each reaction. Additionally, the parameters and trends computed by our study were found to be in agreement with experimental literature.
Reaction pathways associated with Asparagine deamidation.

Reaction pathways associated with Asparagine deamidation.


COVID Therapeutics - From Discovery to Super-Accelerated CMC Development: COVID Therapeutics - From Discovery to Super-Accelerated CMC Development
04:30pm - 06:30pm 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; Michaela Wendeler, Organizer, AstraZeneca PLC; Peter Tessier, Presider, ‍ ; Michaela Wendeler, 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

The COVID-19 pandemic has impacted many aspects of life around the world. It has also initiated an unprecedented race to develop therapeutics and vaccines, necessitating paradigm shifts in drug development. For this session, we invite contributions that highlight the development of innovative COVID therapeutics, especially focusing on solutions for accelerated process development and fast-tracked CMC strategies. We also welcome case studies that describe challenges and technical advances for developing different biotherapeutic modalities, as well as presentations that highlight the scientific, regulatory, and business decisions associated with the pandemic pace. Of interest are strategies that addressed the early need for accelerated drug development, discovery and translation to the clinic, as well as approaches for process characterization, control strategy definition, and process validation. Finally, we welcome presentations highlighting innovative technologies and CMC strategies developed in the face of the pandemic that can likely be leveraged for future development of biotherapeutics in ‘normal’ times.

Tuesday
Facile directed evolution of potent neutralizing nanobodies against SARS-CoV-2
04:30pm - 04:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
SARS-CoV-2 continues to be a worldwide threat to human health, and despite recent advances in therapeutic and vaccine development, prevention and treatment options for the virus remain limited. Methods for the rapid generation of potent neutralizing antibodies are urgently needed. We demonstrate a simple approach for the in vitro isolation of high-affinity neutralizing antibodies from a synthetic nanobody library. We demonstrate that swapping the complementarity-determining regions (CDRs) between low affinity lead clones produces large increases in both affinity and neutralization activity. A nanobody that is affinity matured in this manner demonstrates greater neutralization activity towards the live virus than a potent neutralizing human antibody isolated after infection (CB6) and a potent neutralizing nanobody isolated after immunization (Ty1). We further demonstrate that systematic CDR swapping can be incorporated into the initial sorting process through simple PCR-based methods, allowing identification of high affinity, potent neutralizing nanobodies without the need for initial lead clone selection and subsequent affinity maturation. We expect that these methods will have broad impact in engineering neutralizing nanobodies against SARS-CoV-2 and emerging variants.
Tuesday
AI-enabled deep mutational scanning of interaction between SARS-CoV-2 spike protein S and human ACE2 receptor
04:50pm - 05:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Diwakar Shukla, Presenter, University of Illinois at Urbana-Champaign
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The rapid and escalating spread of SARS-CoV-2 poses an immediate public health emergency. The viral spike protein S binds ACE2 on host cells to initiate molecular events that release the viral genome intracellularly. Soluble ACE2 inhibits entry of both SARS and SARS-2 coronaviruses by acting as a decoy for S binding sites, and is a candidate for therapeutic and prophylactic development. Deep mutational scans is one of the approaches that could provide such a detailed map of protein-protein interactions. However, this technique suffers from several issues such as experimental noise, expensive experimental protocol, and lack of techniques that could provide second or higher-order mutation effects. In this talk, we describe an approach that employs a recently developed platform, TLmutation, that could enable rapid investigation of sequence-structure-function relationship of proteins. In particular, we employ a transfer learning approach to generate high-fidelity scans from noisy experimental data and transfer the knowledge from single point mutation data to generate higher-order mutational scans from the single amino-acid substitution data. Using deep mutagenesis, variants of ACE2 are identified with increased binding to the receptor binding domain of S at a cell surface. We employ the information from the preliminary mutational landscape to generate the high order mutations in ACE2 that could enhance binding to S protein. We also investigate the designed proteins using distributed computing approaches to understand the underlying physics of the spike protein and ACE2 interaction.
Tuesday
Accelerated lead selection and developability assessments for AstraZeneca’s SARS-CoV-2 long acting antibody (LAAB) combination for the prevention and treatment of COVID-19
05:10pm - 05:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Valeria Riguero, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
As a result of the COVID-19 pandemic and its dramatic impact on life around the world, biopharmaceutical R&D laboratories were tasked with discovering and developing monoclonal antibodies (mAbs) using novel strategies on highly accelerated timelines. With this urgent need in mind, AstraZeneca streamlined the typical drug discovery and development activities in an effort to bring COVID-19 antibody therapies to patients as quickly as possible while ensuring that the mAbs met the highest standards of potency, developability and stability. In this presentation, we describe the seamless collaboration between our antibody discovery and protein engineering (ADPE) group and our early stage bioprocess development (BPD) group. The unified team effort characterized 60 potential mAb candidates in less than 7 weeks. Following expression and purification, the mAbs were tested in SARS-CoV-2 in a pseudo-virus neutralization assay, epitope binning assay and in silico sequence liability assessments were performed to narrow the mAb candidates to the top 10 mAbs. The selection of the top two antibodies to enter clinical development was determined by epitope mapping experiments to ensure that the mAbs bound to distinct, non-overlapping regions on the receptor binding domain of the Spike protein and ranking the developability of the mAbs using a combination of techniques including platform expression and purification, liquid stability, analytical method fit, and coformulation assessments. The activities described in this work, performed under pandemic restrictions and lockdowns, resulted in the identification of two highly potent mAbs and enabled their progression to full clinical development in 11 weeks.
Tuesday
Speed-to-clinic and phase I clinical development of a monoclonal antibody therapeutic during the COVID-19 pandemic
05:30pm - 05:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
With therapeutic monoclonal antibodies (mAbs) becoming a predominant class of new drugs developed in recent years, there is an increased emphasis on speed-to-clinic for such medicines that can address unmet clinical needs. As a result, a major focus area for the biopharmaceutical industry has been the shortening of product development timelines to bring a molecule to the clinic for the first time. By using platform technologies and strategies, the process and product development teams at biopharmaceutical companies have been able to significantly reduce the Phase I production timelines for mAbs.

Despite the streamlining of workflows and implementation of platform strategies, an unprecedented, global event like the COVID-19 pandemic can pose severe, unforeseen constraints and challenges to the development timeline of a mAb. Occurrence of the COVID-19 pandemic in 2020-21 warranted several changes in the overall approach to the Phase I development of a mAb to ensure minimal impact to overall project timeline.
This talk will discuss the downstream development of a mAb through the COVID-19 pandemic. Specifically, the work will demonstrate the acceleration and execution of process development activities while navigating the associated risks and challenges, both technical and logistical, presented by the pandemic and responding quickly to changes. Examples of challenges and considerations encountered during development include: a) technical issues such as disulfide reduction and host-cell protein clearance impacting polysorbate excipient stability, b) supply-chain issues affecting the on-time delivery of raw materials (e.g. resins and filters) and subsequent impact on process development and manufacturing, and c) logistics of carrying out end-to-end development & manufacturing in-house vs. outsourcing and additional considerations for the latter. Finally, we will discuss the overall approach, risk mitigation strategies and the lessons learnt that can be applied to future molecules undergoing Phase I development.

Tuesday
Ensuring viral safety for a Covid-19 antibody cocktail
05:50pm - 06:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Kang Cai, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
An antibody cocktail, produced by CHO cells in a fed-batch process, was rapidly developed to combat the COVID-19 pandemic. To ensure the product was not contaminated with endogenous or adventitious viruses in cell culture, the purification process was designed to have multiple, orthogonal, and robust viral clearance steps including virus inactivation, virus filtration and anion exchange chromatography. To support unprecedented tight timelines, the purification process was evaluated for viral clearance capacity based on historical in-house data of similar products and published scientific knowledge. This evaluation, also known as a modular viral clearance claim (as suggested by 1997 FDA Point to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use), led to an estimated ~16 log10 (1016) of cumulative viral clearance capacity from three steps. Regulatory agencies in US and Europe accepted this approach as adequate viral clearance assurance for drug product to be evaluated in clinical trials. Subsequently, product-specific experimental viral clearance studies were performed. The project-specific data confirmed and significantly exceeded the virus clearance estimates by 102.7 determined by the modular claim. The talk will speak to the holistic set of considerations and lessons learned that render this approach a feasible option for regulatory submissions in case of the need for accelerated development of similar products in the future.
Tuesday
New approaches for accelerated biotherapeutics’ development to address a global pandemic
06:10pm - 06:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Kelly Wilson, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
From the start of the COVID-19 pandemic, monoclonal antibodies directed against the coronavirus spike protein were recognized as potentially life-saving therapies. To bring them to the clinic and potential emergency use with unprecedented speed, conventional development timelines had to be replaced by agile strategies that combine technological advances, highly accelerated workflows, and the acceptance of higher business risks.

We present a case study that illustrates how the classical development stages of process development, optimization, and characterization were condensed into one streamlined process. We illustrate how mechanistic modelling enhanced development activities to ensure process robustness. Finally, leveraging prior knowledge was profitably employed in the design of studies and for defining the control strategy.

Upstream Processing: Synthetic Biology & Genome Engineering: Synthetic Biology & Genome Engineering
07:00pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Tara Deans, Presider, ‍ ; Siguang Sui, Presider, ‍ ; Dr Arpan Bandyopadhyay, Presider, ‍ ; Peng Xu, Presider, Guangdong Technion-Israel Institute of Technology Deptartment of Chemical Engineering
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Synthetic biology and genome engineering represent the most fascinating areas in engineering biology. They offer innovative approaches to engineer new biological systems or reprogram existing ones for various applications. By harnessing these tools, we have been able to discover new drug molecules, detect viruses, engineer efficient microbial cell factories to produce high value chemicals, encode decision-making functions, store digital information as well as cure genetic diseases inside living cells. Combined with genome evolution and high throughput phenotyping, we are rapidly expanding our horizon and translating the applications across molecular, cellular or multi-species levels. Further, the progresses made in the fields of synthetic biology and genome engineering have advanced our basic understanding of the complex traits in microbial, plant, and mammalian cells. Talks within this session will highlight the rapid advances in the fields of synthetic biology and genome engineering with a focus on synthetic biology tools development, genome design and evolution, multiplexed gene editing, rapid strain engineering, transcriptional reprogramming, biosensors, engineering of unnatural species, and applications to biotherapy, diagnostics, environment, medicine, and chemical industry.

Tuesday
Directed evolution of terpene synthases for the production of phosphatase inhibitors
07:00pm - 07:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Ankur Sarkar, Presenter, University of Colorado Boulder; Jerome Fox, University of Colorado, Boulder
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Terpenoids are a large, structurally diverse class of medicinally relevant natural products. Despite their role as a longstanding source of pharmaceuticals, however, the chemical complexity and poor natural abundance of these molecules has slowed the development of new terpenoid-based drugs. Systematic modifications to terpenoid-synthesizing pathways can produce diverse sets of terpenoid scaffolds in genetically tractable microbial hosts, but complimentary methods for identifying molecules with important biochemical activities remain largely absent. In this presentation, I will describe an approach for evolving terpene synthases that produce molecules with elusive biochemical activities. My work focuses on a therapeutically relevant objective: the inhibition of protein tyrosine phosphatase 1B (PTP1B), a target for various metabolic diseases and cancers. We encoded this objective into a strain of Escherichia coli and used that strain to evolve terpene synthases that produce PTP1B inhibitors. This method yielded enzyme variants with novel product profiles, enhanced titers, and/or reduced cellular toxicities. Their terpenoid products, which we characterized with detailed biophysical studies, appear to target an allosteric site on the C-terminus of PTP1B. This presentation frames a new approach for using engineered microbial hosts for the evolution of targeted, biologically active metabolites.
Tuesday
Withdrawn
07:20pm - 07:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Tuesday
Elucidation of sequence-function relationships for an improved biobutanol in vivo biosensor
07:40pm - 08:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Nancy Kim, Presenter; Riley Sinnott; Nicholas Sandoval, Tulane University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Transcription factor (TF)-promoter pairs have been repurposed from nature to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF-promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. Sort-seq provides a rational and less labor-intensive strategy to engineer user-defined TF-promoter pairs using FACS and deep sequencing methods. Based on the designed promoter library’s distribution characteristics, sort-seq elucidates sequence-function interactions between DNA-protein. There has been industrial interest in butanol as a renewable fuel. Biobutanol production from renewable feedstocks has been demonstrated, but the production yield remains economically unviable. High-throughput screens on non-growth-related phenotypes and dynamic butanol-dependent regulation represent powerful metabolic engineering strategies that are largely unavailable to these efforts. This capability gap is due to a lack of TF-promoter pairs with user-defined controls. A butanol-responsive TF, BmoR, and its cognate promoter, pBMO, have been previously described in the native form, but pBMO remains relatively uncharacterized and optimizing its function by sequence modification has not been attempted.

In this work, we demonstrate the engineering of the pBMO promoter at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range, and to generate synthetic promoters. We use sort-seq to study the sequence-function relationship of pBMO. A mutagenized pBMO library cloned upstream of gfp in E. coli was induced with butanol and sorted into activity-based (i.e., fluorescence-based) populations. These populations were subsequently deep sequenced and their pBMO mutations correlated with changes in gfp expression, enabling construction of synthetic promoters with desirable characteristics. Identified pBMO mutations were constructed by site-directed mutagenesis and tested in E. coli. Binding characteristics of BmoR-pBMO mutants were confirmed by surface plasmon resonance. The best mutant promoter demonstrated over 4-fold increase in dynamic range over the wild-type. Sort-seq identified essential sites required for proper function of the biosensor and those that increase the sensor output. We also discuss progress toward implementing this biosensor in butanol producing gram-positive solventogenic bacteria.

Tuesday
Cell-free protein systems from Yersinia pestis strains are productive and growth temperature dependent
08:00pm - 08:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Cellular lysates capable of transcription and translation have become valuable tools for prototyping and running genetic circuits, screening engineered functional parts, and producing biological components. Despite the increasing use of lysates, the diversity of organisms used to produce lysates trails behind the diversity of organisms studied and manipulated for biotechnological pursuits. Here we report that lysates derived from Yersinia Pestis CO92-, Kim6+, and EV76 are functional and when driven by σ70 or T7 promoters produce GFP at levels comparable to similar systems in E. coli. Due to the unique natural lifestyle of Y. pestis, lysates were produced from Y. pestis grown at 21°C, 26°C, and 37°C. The lysates generated from Y. pestis grown at 21°C resulted rapid production of GFP followed closely by the 37°C lysates regardless promoter dependency. Together this data demonstrates that, without genetic enhancements, cell-free extracts from Y. pestis are functional and the robustness of the lysates is dependent on the strain as well as temperature in which the bacterium was cultured.
Tuesday
Model-guided engineering of DNA sequences with predictable site-specific recombination rates
08:20pm - 08:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Qiuge Zhang, Presenter, UNIVERSITY OF MINNESOTA; Samira Azarin; Casim Sarkar
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Site-specific recombination (SSR) is an important tool in genome editing and gene circuit design. However, its applications are limited by the inability to simply and predictably tune SSR reaction rates across several orders of magnitude. Facile rate manipulation could in principle be achieved by modifying the sequence of the DNA substrate (rather than the recombinase), but the design principles for rationally doing so have not been elucidated. To enable predictable tuning of SSR reaction dynamics as a function of DNA sequence, we developed an integrated experimental and computational method to parse individual nucleotide contributions to the overall reaction rate, which we used to analyze and engineer the attP DNA sequence for the inversion reaction mediated by the serine recombinase Bxb1. A quantitative polymerase chain reaction (qPCR) method was developed to measure the relative Bxb1 reaction rate in vitro. Then, attP libraries were designed, selected, and sequenced to inform a machine-learning model, which revealed that the Bxb1 reaction rate can be represented by additive contributions of nucleotides at key positions. Finally, we used the model to predict the performance of DNA site variants in reaction rate assays, both in vitro and in Escherichia coli, with flipping rates ranging from 0.01- to 10-fold that of the wild-type attP sequence. Our high-throughput, data-driven method for rationally tuning SSR reaction rates through DNA sequence modification enhances mechanistic insights of recombinase function and expands the synthetic biology toolbox.
Tuesday
Toward a thermally self-regulating living material
08:40pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Engineered living materials (ELMs) retain desirable characteristics of the living component, such as exponential growth, self-repair, and responsiveness to external stimuli. Escherichia coli are a promising constituent of ELMs because they are very tractable to genetic engineering.

Variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment. E. coli experience maximal growth near 37°C. In addition, E. coli protein synthesis decreases below 37°C, while protein misfolding and aggregation tends to increase with temperature.

Here, we develop a genetically encoded mechanism for autonomous temperature homeostasis in ELMs containing E. coli by engineering circuits that change expression of a light-absorptive chromophore in response to changes in temperature. Our simulations show that by increasing absorptivity below 36°C, the material will heat above the ambient temperature to preserve optimal growth and protein expression, and thus material functionality.

We program bacteria to respond to temperature using temperature-sensitive transcriptional repressors (TSRs). Two families of TSRs with switching temperatures ranging from 36°C to 44°C have been developed in our lab by directed evolution of TcI, a temperature-sensitive mutant of bacteriophage λ repressor cI, and TlpA, a transcriptional auto-repressor from the virulence plasmid of Salmonella typhimurium. These thermal bioswitches can be further tuned for optimal switching in the ELM application.

Formation of a black chromophore from a pale yellow precursor is enzymatically catalyzed. Integrating the gene for this enzyme into a genetic circuit with a down-shifted mutant of TlpA enables E. coli to express black chromophore at low temperatures and not at high temperatures.

We measure the ability of patches of E. coli (simulating an E. coli-based ELM) to grow in a custom lighted incubator at different ambient temperatures by observing patch diameter and thickness. We continuously monitor E. coli temperature under illumination using thermal IR imaging with custom controller software. Comparison of experimental results and simulations will be presented. We demonstrate thermal control of pigmentation and resulting increase in sample temperature.

Downstream Processing: in Silico & Mechanistic Modeling of Downstream Bioseparations: In Silico & Mechanistic Modeling of Downstream Bioseparations
07:00pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Gunnar Malmquist, Presider, Cytiva; Dr. Camille Bilodeau, Presider, Massachusetts Institute of Technology
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Mechanistic models capable of describing bioseparations have long been available but have not yet managed to break into mainstream biopharmaceutical development. This is rapidly changing as the digital revolution is sweeping through the biopharmaceutical industry, resulting in new, computational workflows that can be readily integrated with modeling to achieve smart and disruptive downstream process development. The session invites speakers from all fields to share their advances and case studies in modeling of bioseparations. In particular, we invite speakers to share their advances in emerging modeling applications including modalities beyond mAbs, non-chromatography steps (filtration, viral clearance, etc.), and multi-step modeling. We also encourage submission of research involving hybrid modeling approaches such as statistical and mechanistic modeling synergies, models that leverage protein sequence/structure, models that utilize developability/manufacturability data, and molecular modeling approaches (fundamental studies, predictive models, or hybrid molecular/mechanistic models). Finally, we invite research that addresses key practical considerations for mechanistic models in bioseparations including minimizing mechanistic modeling hurdles (resources, time, complexity, or analytical burden), assessing and improving model accuracy, transferring models as part of tech transfer and scale-up, or using models for regulatory filings or lifecycle management.

Tuesday
Optimization and characterization of multimodal chromatographic processes using mechanistic models derived from high-throughput batch isotherm data
07:00pm - 07:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Scott Altern, Presenter, Rensselaer Polytechnic Institute; Jessica Lyall; Chris Williams; John Welsh; Jamie Peyser; Vijesh Kumar; Abraham Lenhoff; Steven Cramer, Rensselaer Polytechnic Inst
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In recent years, high-throughput batch and column screening (HTS) have seen widespread use in the development of chromatographic processes and have been presented as a solution to mitigate bottlenecks in process development. Moreover, mechanistic column modeling has been shown to be a viable option to reduce process development time, while also enabling straightforward development of processes for enhanced impurity removal and assurance of process robustness.

In this work, we present a streamlined workflow to integrate generation of high-throughput batch isotherm data with mechanistic column model development. The developed models are then employed to create processes for product variant removal and control as well as for characterization of the chromatographic design space. Here, we apply this strategy to monomer-aggregate separations and simultaneous characterization of acidic and basic charge variants for a mAb using a multimodal cation exchange resin. Further, we demonstrate the flexibility of the mechanistic model for predicting chromatographic behavior on systems of varying scale, such as RoboColumns for process characterization and larger scale lab columns for process development. The process characterization studies are also used to inform the unique dependence on pH and ionic strength that can occur in these complex systems and their impact on product variant removal. Finally, we demonstrate how this workflow can be readily employed for other modes of operation, such as cation exchange chromatography.

Tuesday
Impurity behavior in flow-through ion-exchange chromatography
07:20pm - 07:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The identity, quantity, and persistence mechanism of individual impurities in flow-through ion-exchange (IEX) are often unknown. This, coupled with an incomplete understanding of resin retention differences, necessitates resin screening during process development. An improved understanding could help expedite development, reduce costs, and increase consistency with quality by design principles. For this reason, an investigation is reported here of contributions to inadequate impurity clearance in flow-through IEX. Transport contributions are examined using column simulations, suggesting a dependence of breakthrough volume on the particle Péclet number. This dependence is explored experimentally, and the implications are examined. Thermodynamic information in the form of retention factor–ionic strength data are also compiled from experiments and literature, providing data for over 200 protein-pH-resin combinations. Fitting these data reveals a correlation between retention strength and the characteristic charge. The correlated data may serve as a heuristic when studying host-cell proteins (HCPs), for which experimental data are often unavailable. This heuristic’s utility is investigated using proteomic analysis of HCPs found in the protein A eluate of an antibody manufacturing process.
Tuesday
Impact of multimodal ligand density on free energy of adsorption of proteins to ligand-immobilized SAM surfaces: A molecular dynamics study
07:40pm - 08:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Mayank Vats, Presenter; Shekhar Garde; Steven Cramer, Rensselaer Polytechnic Inst
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Multimodal chromatography presents unique selectivity in bio-separations, but a fundamental understanding of differences in multimodal (MM) ligands’ behavior still eludes us. Recent simulations work in our lab on MM-ligand immobilized self assembled monolayer (SAM) surfaces has demonstrated that ligand flexibility and density differences can result in surfaces with relatively large patches of hydrophobicity and charge. These self-associated ligand patterns have also been found to be persistent in chromatographically relevant higher salt concentrations and could govern protein-surface interactions. Here we develop that premise using enhanced sampling in molecular dynamics simulations of these systems. First, INDirect Umbrella Sampling (INDUS) was used to study the hydrophobic nature of surfaces where immobilized MM-ligands were found to aggregate and create hydrophobic and charged patches, and then compared with low ligand density surfaces without ligand self-association. To further understand the importance of ligand clustering in protein adsorption, free energies of binding of model proteins to a series of SAM surfaces with varying MM-ligand densities were calculated from potential of mean force (PMF) measurements performed using umbrella sampling. The possibility that water-mediated interactions during binding and unbinding at the protein-SAM interaction interface show hysteresis-like behavior was also investigated. Correlations between the calculated free energies and corresponding patch distributions for those ligand surfaces, inform us about the role of ligand density driven self-association in adsorption. Our study presents an understanding of hydrophobic behavior in multimodal systems at different ligand densities and connects multimodal resin surface properties with density dependent behavior during protein adsorption, which in turn has implications for surface property driven multimodal resin design and selection.
Tuesday
Affinity chromatography predictive scaling using miniature Fibro units and mechanistic modeling
08:00pm - 08:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Affinity chromatography such as Protein A affinity chromatography is an important step in the purification of monoclonal antibodies (mAbs) and mAb-derived biopharmaceuticals. While the biopharma industry has extensive expertise in the operation of Protein A chromatography, the mechanistic understanding of the adsorption/desorption processes is still limited and scaling up and scaling down can be challenging. Benner et al. (2019) describes mass transfer effects in bead-based resins as one possible explanation for the latter. With the advent of convective media such as Fibro technology, complex mass transfer and pore diffusion technologies are about to go obsolete. Fibro technologies drastically simplify the application of mechanistic modeling, since only the macroscopic fluid dynamics of the Fibro unit and the thermodynamic binding effects are left to be elucidated.
In a collaborative study, we performed extensive experimental campaigns with varying cycles and mAb feedstock. The results form the basis for mechanistic modeling of the mAb adsorption and elution behavior, leadingto a predictive pH-extended stoichiometric model. Although only few runs were performed to develop the model, the simulation results provide accurate predictions for varying scales with high accuracy
In summary, the results show how fiber-based Protein A systems and processes can be developed quickly with limited material expenditure and how reliable scale-up and scale-down can be predicted with high accuracy using a mechanistic model.

Tuesday
Applications of mechanistic/hybrid modeling in bioprocess development and scale up
08:20pm - 08:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
To realize the first-mover’s advantage and maximize the market uptake, pharmaceutical companies are hard at work to significantly reduce product development cycles. Among all the initiatives that are often considered for an accelerated timeline, implementation of digital technologies has witnessed a surge over the past few years. A robust digital foundation not only helps with faster product delivery, but also significantly reduces financial burdens. While digital technologies include a wide variety of tools, herein, we turn our attention to the application of “mechanistic modeling” in bioprocesses. It is widely accepted that among all the modeling strategies for bioprocesses, “mechanistic modeling” and the use of first principles is a first go.
A quick glance at a typical process for the development of biotherapeutics reveals that the existence of fluid flow is a common factor among all the unit operations. Therefore, an in-depth understanding of hydrodynamics is key for bioprocess development. To achieve this, first principle mathematical approaches can provide a reliable path to understanding fluid flow mechanics. Additionally, first principle mechanistic models can often be coupled to allow for the inclusion of multiple biophysical/biochemical phenomena and the generation of a holistic modeling strategy for a specific unit operation. In this presentation, we share some applications of mechanistic/hybrid modeling in bioprocess development and scale up.

Tuesday
Withdrawn
08:40pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Protein Aggregation & Immunogenicity : Protein Aggregation & Immunogenicity
07:00pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Cesar Calero Rubio, Organizer, Sanofi Genzyme; Mary Krause, Organizer, Bristol Myers Squibb; Krishna Mallela, Organizer, Univ of Colorado Denver; Kayla Sprenger, Presider, ‍ ; Daniela Verthelyi, Presider, ‍ ; Cavan Kalonia, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Protein aggregation and particle formation pose substantial challenges during the development of new biotherapeutics due to their potential for eliciting undesirable and harmful immune responses. This session will focus on recent advances from both academia and industry to characterize the link between protein aggregates and immunogenicity, towards improving mitigation and control strategies. Suggested topics include, but are not limited to: 1) new approaches for understanding the molecular mechanisms of protein aggregation and aggregation mitigation strategies at both the formulation and bioprocessing stages (e.g. assessing conformational and colloidal stability due to changes in pH or chemical modification of biotherapeutics), 2) methods to characterize visible and subvisible protein aggregates; 3) new methods for predicting protein aggregation tendencies; and 4) approaches to correlate and predict the impact of protein aggregates on immunogenic responses. Computational and experimental (in vitro, in vivo, or clinical) approaches are welcome.

Tuesday
Withdrawn
07:00pm - 07:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Tuesday
Utilization of predictive models to assess therapeutic antibody aggregation
07:20pm - 07:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Paria Ghannadian, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Protein aggregation in therapeutic antibody products can potentially jeopardize product shelf life, efficacy, and patient safety. In order to prevent undesired protein aggregation, the colloidal stability of protein therapeutics is optimized over the course of drug product formulation screening. However, direct characterization of aggregation propensity over long-term stability studies during formulation development can be time-consuming and labor-intensive. Therefore, it is advantageous to expedite stability studies through initial assessment of underlying protein biophysical parameters that relate to aggregation propensity, and the application of said values within corresponding predictive models. In this study, 27 commercial monoclonal antibodies (mAbs) consisting of immunoglobulin 1, 2, and 4 subclasses with isoelectric point values between 7 and 9 were formulated into 5 different aqueous buffers and assessed for colloidal stability over 12 weeks at 45 Celsius (long-term forced degradation conditions). Additionally, protein-protein interaction was evaluated through measurement of biophysical parameters (i.e. diffusion interaction parameter (kD); Kirkwood Buff Integral (-G22)), and effective solution charge (z*). Biophysical parameters and solution charge were shown to statistically correlate with the aggregation propensity of all antibody-buffer combinations. It was observed that buffers with low pH/ionic strength (i.e. sodium acetate; L-histidine hydrochloride) resulted in higher (positive) values for kD, -G22, and z*; ultimately, this translated to reduced protein aggregation, which was attributed to predominant electrostatic repulsive forces between the solvated protein species. Finally, with the implementation of a predictive model using maximum entropy theory principles, the probability of protein aggregation could be estimated through measurement of kD, G22, and z* to facilitate preformulation and formulation development workflows and minimize user reliance on broad long-term stability studies.
Tuesday
Using high-pressure, sub-zero conditions to gain insights for mechanisms of monoclonal antibody unfolding and aggregation
07:40pm - 08:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Aggregation of therapeutic proteins is a well-known issue that can occur during multiple steps of manufacturing and storage. Experimental models of aggregation under cold (sub-zero °C) storage conditions are often confounded because of the presence of ice, which is also a potential denaturing agent via adsorption of proteins to the ice-water interface. Hydrostatic pressure can be used to prevent water from crystallizing at sub-zero temperatures, allowing for the characterization of protein structural perturbations leading to aggregation in solution, and separating those effects from aggregation at bulk interfaces.
The effects of incubating two different monoclonal IgG1 antibodies (MAb) at high pressure (up to 350 MPa) and low temperature (0 to -20 °C) were examined in this work. Intrinsic fluorescence and small-angle neutron scattering (SANS) were used to observe the in situ effects in an effort to not only characterize tertiary structure at these conditions, but also to detect aggregation prone intermediates that are often difficult to characterize. In both cases but to varying extents, partial unfolding of the MAbs was observed under a range of pressure/ temperature conditions. Fourier Transform infrared spectroscopy (FTIR) was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubations at sub-zero temperature under pressure was independent of reversibility as determined by assessment of tertiary structure. Several pressure and temperature conditions were also used to discern the respective contributions of the isolated MAb fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed unique partial unfolding profiles and reversibility, indicating a complex relationship to full MAb unfolding and aggregation behavior in the case of both antibodies. The combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments. This work may ultimately lead to better prediction of aggregation propensity during long term storage and faster screening of new stabilization strategies for MAb therapeutics.

Tuesday
Impact of therapeutic mAb aggregation on its biological activity
08:00pm - 08:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Dr Rozaleen Dash, Postdoctoral Fellow, Presenter, Indian Institute of Technology Delhi; Rohit Bansal; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Monoclonal antibody (mAb) products are known to be prone to molecular instabilities like aggregation, fragmentation, oxidation, and reduction. Aggregation is a major issue for the advancement of marketed biotherapeutics products which can induce adverse immune responses in patients that may affect safety and efficacy. Though therapeutic formulations are designed to stabilize the product and minimize product degradation, aggregation is unavoidable and continues to be a concern for manufacturers of protein based therapeutic products. Aggregate particles of various sizes and morphologies are formed when subjected to physical, mechanical, and/or chemical stresses. These stresses may be experienced during manufacturing, storage, filling, formulation development and shipping. While there is widespread consensus that protein aggregation can enhance immunogenicity, the underlying immunological and biological mechanisms are not completely understood. A better understanding of protein aggregation can be gained by developing robust analytical techniques for monitoring level as well as nature of the aggregates. This study investigates how mAb aggregates generated by a variety of mechanical, thermal and chemical stresses impact the biological activity of a biotherapeutic. Increased aggregation resulted in a decrease in biological activity, as confirmed by cell based assays such as antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) and ligand binding assays such as surface plasmon resonance (SPR). It was observed that aggregates formed due to extreme pH (pH 3.5 and pH 11.0), stirring (1 d stir and 3 d stir), thermal stress, and oxidation via CuSO4 have the most impact on potency of the therapeutic. In contrast, aggregates formed due to stresses from pipetting, milder pH (4.3 and 8.5), oxidation via H2O2, freeze-thaw (Ft-slow and Ft-fast) have relatively less impact on the potency of the mAb biotherapeutic. The results affirm that understanding of the mechanism of aggregation is critical for achieving consistent product quality and the resulting efficacy.
Tuesday
Analytical platform for monitoring aggregation of monoclonal antibody therapeutics
08:20pm - 08:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Rohit Bansal; Surbhi Gupta, Research Scholar, Presenter, Indian Institute of Technology Delhi; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Purpose To develop an analytical platform for the estimation as well as characterization of aggregates over the complete size spectrum (from invisible monomer to visible precipitates).
Methods Two mAb samples were incubated at 30°C in dif- ferent buffer systems of protein A chromatography for observ- ing degradation due to aggregation. The aggregation in these samples was quantified by size exclusion chromatography (SEC), dynamic light scattering (DLS), and micro flow imag- ing (MFI).
Results Theresultsobtainedfromvariouscharacterizationtools were analysed in various size ranges - size exclusion chromatog- raphy (SEC) (1 nm - 25 nm), dynamic light scattering (DLS) (10 nm - 5 μm), and micro flow imaging (MFI) (2 μm - 300 μm). Since each characterization tool covers a particular size range, data from multiple tools was collected in the “hand- over” regions to demonstrate accuracy of the platform. Conclusions Based on the observations from the experiments, an analytical platform has been proposed covering the whole size spectrum that would be of utility to those engaged in formulation development as well as other aspects related to stability of biotherapeutic products.

Tuesday
Impact of mAb aggregate on the capacity and efficiency of chromatography process
08:40pm - 09:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Monoclonal antibody (mAb) aggregation is one of the most critical risk factors for safety and efficacy of the therapeutics. However, aggregates are generated easily during manufacturing and storage steps and continue to be a major problem in downstream process development. Separation of soluble aggregates is most challenging when the aggregates have surface chemical properties similar to the monomer that can interact with resin including affinity chromatographic resin. In this presentation, we will show the impact of aggregate on the capacity and efficiency of chromatography steps such as protein A, ion exchange or HIC (hydrophobic interaction chromatography) columns. As titer from upstream increases, capacity of the resins is more of importance and impurities such as aggregate reduces the binding capacity of monomer significantly. We will also demonstrate efficient removal of mAb aggregates by utilizing additives and highly selective chromatography media which results in increase in process throughput and product purity.