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Downstream Processing: Membrane-Based Downstream Bioprocessing: Membrane-Based Downstream Bioprocessing
02:00pm - 03:40pm 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; Mahsa Hadidi, Presider, ‍ ; Ranil Wickramasinghe, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

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

Tuesday
Enhancing productivity: High-throughput antibody purification in minutes
02:00pm - 02:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Antibodies are purified at small scale in many different applications: In the antibody discovery, engineering, and characterization businesses, high purity antibodies are needed to minimize background interference for more accurate assessment. In antibody sequencing, a small quantity of antibodies of moderate purity is needed for subsequent processes. Many antibody sequencing companies also need to provide clients purified antibody at the milligram scale along with sequencing reports. For cell line development, supernatants are usually purified to understand the levels and types of impurities, such as the HCP profile, and to determine other properties of the antibodies. Purifications for each of these activities can be laborious, time consuming, and inhibit productivity. Presented here are antibody purification case studies using two new rapid, high-throughput antibody purification tools. These tools are based on Purilogics’ Protein A membrane adsorbers and can perform purifications in minutes, not hours. One of the tools is a multi-well Protein A membrane plate. Compared to magnetic bead-based technology, the Protein A membrane plates are faster, easier-to-use, more economical, and require no time-consuming incubations. Alternative offerings include a syringe filter-like Protein A membrane column, which enable purifications without a FPLC in just a few minutes. These case studies highlight novel strategies to significantly increase discovery stage antibody output.
Tuesday
Mechanistic modelling-based optimization and scale-up of ion-exchange membrane chromatography
02:20pm - 02:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Jan Hedrich, Presenter, Hochschule Mannheim University of Applied Sciences; Rita Steigmiller; Romas Skudas; Christian Dr. Frech
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Manufacturing complex biopharmaceuticals with higher efficiency and simultaneously reducing costs sets pressure on downstream processing and encourages the development of novel techniques. Membrane chromatography, a relatively new technique, holds excellent potential in the purification of proteins. Its primary purpose of being operated at very high flow rates has been combined with functionality that rivals particle-based materials in efficiency and flexibility. Usually operated in flowthrough mode, the applications of membrane chromatography in industrial processes range from the removal of contaminants like DNA and HCPs to the purification of large biomolecules like viruses, VLPs, and protein complexes.
This study aims to demonstrate the usability of cation-exchange membrane chromatography in a bind and elute mode. As an example, a high-resolution separation of monoclonal antibody charge variants was used. After calibration of a mechanistic membrane chromatography model, an in-silico simulation strategy was applied to optimize the separation of mAb charge variants. Additional data from high loading studies (up to 35 mg/mLMV) was included to enable simulations at far higher loadings. Membrane volume and flow rates were successfully scaled up from 0.87 ml to 10 ml membrane volume and from 10 mL/min to 240 mL/min, respectively. For a cost-effective approach, in silico simulations were used to improve and optimize the separation of charge variants using a combination of flowthrough and step elution.
At similar conditions, the novel membrane prototype has a DBC10% of 90 mg/mL, comparable to its resin-based counterpart. The resolutions achieved using the membrane devices are close to those obtained through column chromatography. However, much higher flow rates are possible using the latter. The flow rate's influence is significantly different from that of column chromatography, indicating distinctive mass transfer behaviors.

Tuesday
Withdrawn
02:40pm - 03:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Tuesday
Effects of pressure fluctuation on viral clearance
03:00pm - 03:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Esha Vyas, Presenter, Asahi Kasei Bioprocess
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In virus filtration, which is an integral part of downstream processes for biopharmaceutical products, viruses and other contaminants are effectively removed by nanofiltration. Planova filters are operated in dead-end mode by either constant pressure or constant flow rate control. When strict pressure control is required, Asahi Kasei Bioprocess (AKB) recommends using constant pressure control, either by house air or compressed air tanks. To examine the effects of pressure level and fluctuation during filtration on virus removal, we examined viral clearance for constant pressure control by compressed air (no fluctuation) and constant flow rate control by peristaltic pump with two pump heads (limited fluctuation, range of <4 psi) at three pressure levels: low pressure, just within manufacturer recommend maximum operating pressure (14.2 psi) and high pressure (exceeding manufacturer recommended maximum operating pressure). Additionally, one pump head was used to produce exaggerated fluctuation (range of >9 psi) covering the low and high pressure ranges. A total of 150 mL of 5 g/L BSA with porcine parvovirus (PPV) spike (virus challenge, 10.5 logs TCID50/m2) was filtered on 0.001 m2 Planova 20N filters and collected in two equal fractions for PPV determination. Pressure fluctuation during filtration runs did not cause marked decreases in PPV LRV under any of the conditions tested. Additionally, we will discuss the effects of a gradual pressure increase, or ramp up, particularly at the start of the buffer flush filtration for the product recovery step of a Planova filtration processes.
Tuesday
Proposed standard virus filter validation method: One that covers all worst-case conditions in a single test
03:20pm - 03:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Paul Genest, Presenter, MilliporeSigma
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
This presentation will provide a comprehensive review of the different virus retention behaviors, and mechanisms, for two parvovirus filters. The same conditions that cause virus breakthrough, for these two parvovirus filters, apply to all parvovirus filters. It is only the degree of the impact that differs for each specific filter type. This is explained by the fact that each virus filter has a unique material of construction, membrane morphology, and membrane pore size distribution. This presentation will also review the relative virus retention behaviors of other virus filter types in studies where they were tested all together. The primary objective is to discuss worst case conditions and critical parameters for parvovirus filters in general. This is a very important and timely discussion to have now, with over two decades of experience running these filters. It is important for filter suppliers, filter users, and regulators to have this open discussion and clarify what is and is not critical for any specific virus filter, so the process can be effectively validated and controlled. A proposed option for a single standard validation test method will be described, one that includes all of the virus breakthrough causing conditions in one test. This agreed upon single standard validation test method, with agreed upon reasonable worst-case endpoints, would make the validation process more predictable and efficient, and improve safety when it comes to all virus filter types, and be more transparent for everyone. All would know what needs to be tested and why, and what reasonable worst-case endpoints should be used.
Tuesday
Membrane microfiltration of pathogenic bacteria in individual 500 mm diameter hollow fibers
03:40pm - 03:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Bioburden management in downstream pharmaceutical manufacturing – where the cost of a failed production lot is on the order of $1 million – requires detection of microbial contaminants (Westman, 2017). Methods based on plating, PCR, or immunoassay necessitate sample concentrations equivalent to 100 to 1000 microorganisms/mL in 5 to 50 mL volumes. Hollow fiber microfiltration, preceded by a short enrichment in selective media, achieves these titers in 7 hours and enables detection of one colony forming unit/g initial sample, as demonstrated for egg white and spinach, while enrichment by culture to achieve the same detection limit required 24 hours (Ku et al., 2016, 2017, 2019). A multiplexed hollow fiber microfiltration system developed in our laboratory to concentrate four samples at the same time facilitated systematic studies of interactions of HF membranes with viable bacteria (Zuponcic et al., 2019). This led to research on the effect of shear on recovery of viable bacteria using in-house fabricated, 0.2 µm cutoff, 0.5 mm diameter individual polyethersulfone hollow fibers. Linear velocities of 68 to 424 cm/sec at the fiber entrance were 10 to 60 x, respectively, higher than commercial microfiltration modules. The corresponding flowrates of 8 to 50 mL/min generated shear rates of 10,000-70,000 s-1 at Re = 300 to 2000 and 30 psi. Micrographs of bisected hollow fibers after HF microfiltration of dilute bacterial suspensions showed preferential accumulation of bacteria at HF membrane surfaces within 3 cm of the inlet. Plating confirmed that the test bacteria, Salmonella enterica and GFP-producing Escherichia coli, retained viability despite high shear rates. We report impacts of hydrodynamics on shear-dependent behavior of bacteria on the wall of an individual hollow fiber and application of these observations to enhancing recovery of concentrates of living bacteria for purposes of estimating bioburden or for detecting presence of viable bacteria at the 1 cfu level.
Advances in Process Development & Manufacturing of Biologic Drug Products: Advances in Process Development & Manufacturing of Biologic Drug Products
02:00pm - 04:00pm 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; Sanket Patke, Presider, ‍ ; Anna Schwendeman, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

The space of biologic therapeutics is continually expanding due to the development of new protein, gene therapy and cell therapy formats, new modes of action, new screening technologies, and new design tools. This session will focus on recent advances in drug product process development and manufacturing. Suggested topics include: Scale-down pilot models, computational modeling and digital tools, effects of excipients, freeze and spray drying, suspensions, non-aqueous biologic drug products, aerosols, biosimilars and vaccines manufacturing. The scope of this session will also include the topic of process development pertaining to gene therapy, cell therapy, CART-T, and lipid nanoparticle-based therapeutics.

Tuesday
Justification of small-scale models: An industry perspective
02:00pm - 02:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Dr Robert Luo, Presenter, GSK
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Small-scale models and their qualification are important steps of biopharmaceutical process development, characterization, and validation. Despite being required by regulatory bodies, there is no guidance on current best practices and companies use many different qualification approaches throughout the industry. The design, execution and analysis of SSMQ studies can be challenging due to this lack of clear guidance on current best practices.

It must be experimentally demonstrated that an SSM, running at the appropriate process parameter settings (inputs), has representative performance and quality attributes (outputs) to the manufacturing scale. It is important to understand the degree to which these models represent the commercial process, including any limitations or differences that might exist (e.g. offsets). These factors may have an impact on the relevance of information derived from the models in order to justify the use of SSM to meet the above regulatory expectations.
The procedure for qualifying SSMs should follow defined engineering rules and apply statistical methods when applicable. To this end, the white paper presents current understanding on how to successfully qualify an appropriate and applicable SSM, including design, execution, data analysis and justification of the results. Both upstream and downstream unit operations are covered and supported by case studies. Detailed considerations for designing and executing process characterization studies were considered out of scope for thse white paper.

In order to address the gap in regulatory guidance, BioPhorum’s Development Group’s Qualification of Small-Scale Models Workstream has produced an extensive whitepaper called Justification of Small-Scale Models: An Industry Perspective, which is due to published in April/May 2021. The whitepaper provides options and tools for design, execution, and data analysis of SSMQs together with illustrative case studies. Representatives of the workstream would like to present an overview of the paper during the conference.

Tuesday
Aggregate- and particle-associated host-cell proteins in monoclonal antibody bioprocessing
02:20pm - 02:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Host-cell proteins (HCPs) are process-related impurities that are generated in monoclonal antibody (mAb) production by CHO cells. While the majority of HCPs are removed effectively by downstream purification steps, some may remain even in drug substance, possibly having detrimental effects on drug stability or patient safety. In this study, HCPs in the key downstream process steps of 7 different mAbs, including harvested cell culture fluid (HCCF), protein A eluate, and polishing chromatography pools, have been identified and/or quantified by LCMS/MS in data-independent acquisition (DIA) or data-dependent acquisition (DDA) mode. Of the HCPs identified in the seven protein A pools, 41 HCPs were consistently found in all of the seven samples and interestingly more than a quarter of those HCPs are known to be involved in aggregate formation or protein degradation in mammalian cells. In an effort to understand the origins of those HCPs and to determine possible effects on their persistence through the initial capture step (protein A chromatography), size-exclusion chromatography (SEC) fractions were collected from HCCF and protein A pools and analyzed by LC-MS/MS analysis in DDA mode, which enabled identification of HCPs that may be incorporated into aggregates or clusters. Such association may help explain the persistence of some HCPs in the mAb process stream even in the absence of product association, a role that has previously been postulated for chromatin. The implications of the results for understanding HCP persistence in mAb manufacturing will be discussed.
Tuesday
Addressing challenges in employing advanced manufacturing techniques for lyophilization of biologics: Role of process analyzers and advanced product characterization
02:40pm - 03:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Lyophilization is an old-age technique employed for manufacturing of about 50% of biologics. Unfortunately, there have been little advancement in lyophilization process development, monitoring and control.Thus, there is a need to adopt innovative and advance techniques for lyophilization of biologics. Controlled ice nucleation (CIN) technology is an advanced technology for lyophilization. CIN improves process efficiency, batch homogeneity and reduces process time. However, CIN is yet to be employed at manufacturing scale for lyophilization of biologics. due to business, technical, and regulatory challenges. For this reason, this research was conducted to improve our understanding of CIN, identify potential risk factors and mitigating strategies.Unexpectedly, mass flow analysis of five lyophilization cycles of the same product revealed a direct relationship between the degree of supercooling and primary drying efficiency. The use of an in-line heat flux monitor for product and process monitoring allowed the determination of critical parameters such as the onset of ice-nucleation, the degree and duration of supercooling, percentage of frozen product and adequacy of the freezing hold time. An in-depth analysis of the heat flux data and advanced product characterization data revealed the efficiency of the primary drying was not only dependent on the degree of supercooling but also microstructural changes during primary and secondary drying. AI characterization and simulated of mass flow values from 3D cake images confirmed the dependence of mass flux on microstructural changes post freezing. Thus, a lower degree of supercooling may not always translate to an increased primary drying efficiency but may reduce the risk of collapse and loss of product quality. Further, batch homogeneity was not only dependent on the ice nucleation but also dependent on product changes post-ice nucleation.Thus, adoption of CIN may not necessary improve the inter and intra-batch product homogeneity.More importantly our work shows how a combination of advanced in-line and at-line PAT tools specifically in-line mass flow analyzer, heat flux monitor, automated water activity analyzer and high resolution cake characterization and artificial intelligence analysis, can be employed to improve our understanding of the relationship between CIN process parameters, process performance and the ensuing product quality attributes.
Tuesday
Withdrawn
03:00pm - 03:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Tuesday
Advanced solid-state characterization to understand the impact of protein-excipient interactions on aggregation of spray dried protein solids
03:20pm - 03:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Qi Zhou, Presenter; Yuan Chen; Dr Jing Ling, Presenter, Merck & Co.; Mingyue Li; Yongchao Su; Lynne Taylor, Purdue University; Eric Munson; Elizabeth Topp
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Mannitol, leucine and trehalose have been widely used in spray-dried formulations, especially for inhalation formulations. The effects of these excipients on protein physical stability in spray-dried solids were studied here using bovine serum albumin (BSA) as a model protein. The spray-dried solids were characterized with scanning electron microscopy (SEM), powder X-ray diffraction (PXRD) and solid-state Fourier-transform infrared spectroscopy (ssFTIR) to analyze particle morphology, crystallinity and secondary structure change, respectively. Advanced solid-state characterizations were conducted with solid-state hydrogen-deuterium exchange (ssHDX) and solid-state nuclear magnetic resonance (ssNMR) to explore protein conformation and molecular interactions in the context of the system physical stability. Trehalose remained amorphous after spray drying, and was miscible with BSA, forming hydrogen bonds to maintain protein conformation whereby this system showed the least monomer loss in the stability study. Both crystalline and amorphous forms of mannitol were detected in the spray-dried BSA-mannitol solids by ssNMR, which led to partial loss of stability to BSA. Leucine showed the strongest crystallization tendency after spray drying and did not provide a stabilizing effect due to substantial immiscibility and phase separation with BSA as a result of crystal formation. This study demonstrates the pivotal role of advanced solid-state characterization techniques in understanding the physical stability of spray-dried protein solids.

Tuesday
Raman spectroscopy for in situ, real time monitoring of protein aggregation in lyophilized biotherapeutic products
03:40pm - 04:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 05
Nitika Nitika, Presenter, IIT-Delhi; Hemlata Chhabra; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Quality of biotherapeutic products is of paramount importance for ensuring patient safety. Analytical tools that can facilitate rapid quality assessment of the therapeutic product at the point of care are very much in demand. In this article, we apply chemometrics based analysis of Raman spectra towards quantitative prediction of protein aggregation in lyophilized biotherapeutic products. Two commercially available therapeutic proteins, erythropoietin (EPO) and human growth hormone (HGH), have been used to demonstrate the applicability of the proposed approach. Thermally induced protein aggregation was monitored by size exclusion chromatography as well as Raman spectroscopy with a 785nm wavelength laser. Partial least square (PLS) regression was used to analyse the Raman spectra and create a model for quantitative determination of aggregate. Satisfactory performance was observed with both EPO and HGH with R2 of 0.91 and 0.94, cross-validation correlation coefficient of 0.85 and 0.89, and Root Mean Square Error computed from cross calibration (RMSEcv) of 5.25 and 1.92, respectively. The developed approach can enable rapid and accurate assessment of aggregation in lyophilized samples of biotherapeutic products. The study also demonstrates novel use of Raman spectroscopy for protein quantification through a vial.
COVID Vaccines - From Discovery to the Fastest Vaccine Development in History: COVID Vaccines - From Discovery to the Fastest Vaccine Development in History
02:00pm - 04:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Frank Kotch, Organizer, Pfizer Inc; Varnika Roy, Organizer, GlaxoSmithKline Plc; Dr. Shannon L Servoss, Organizer, University of Arkansas; Avdreas Kuhn, Presider, ‍ ; Kunal Aggarwal, 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

Given the emergence of the global COVID-19 pandemic, a lot of work has been initiated in early 2020 to rapidly develop vaccines against the corresponding virus, SARS-CoV-2. Such a human vaccine is essential initially for the protection of high-risk individuals and health care workers and ultimately to obtain so-called herd or community immunity. Traditional approaches to prophylactic vaccine development (e.g. attenuated strains of viruses, heat-inactivated viruses or recombinant proteins) have lengthy development timelines to establish safety and efficacy. This session will focus on talks that highlight the challenges associated with accelerated vaccine development in the face of a global pandemic and that provide examples of how these have been tackled. The topics solicited include but are not limited to case studies showcasing use of disruptive approaches to accelerate vaccine development, use of platform technologies to develop COVID-19 vaccines under rapid timelines, innovative approaches in addressing long lead times associated with analytical development, characterization and release testing, and process scale-up and manufacturing to meet the demand of billions of vaccine doses.

Tuesday
Rapid development of COVID-19 mRNA vaccines
02:00pm - 02:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Kizzmekia Corbett, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here, we show that mRNA-1273 induces potent neutralizing antibody responses and protects against SARS-CoV-2 infection in the lungs and noses of mice and NHP without evidence of immunopathology. We also detail the immune responses in human from the Phase 1, 2, and 3 clinical trials. mRNA-1273 is currently in a phase III trial and has been deemed safe and effective, with a 94.1% interim phase 3 efficacy rate. To date, millions of people have been vaccinated under an FDA Emergency Use Authorization. Moving forward, we are investigating the utility of mRNA vaccine platforms as global variants arise, assessing immune corelates of protection, and establishing key evidence to estimate durability of mRNA-1273-elicited protection.
Tuesday
Withdrawn
02:40pm - 03:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Tuesday
Manufacturing and design of a COVID-19 vaccine antigen for global access
03:00pm - 03:20pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
To address the COVID-19 pandemic globally, the world will require >10 billion doses of vaccines for SARS-CoV-2. This requirement exceeds the annual capacity for manufacturing vaccines for all other diseases by >2x. Most of these doses are required in low- and middle-income countries (LMICs), where platforms for the manufacturing and distribution of vaccines are limited. To fulfill this need, it is clear that further innovations in vaccine design and manufacturing are required. Protein subunit vaccines are promising candidates for global distribution because they can be manufactured at low costs in microbial hosts. The yeast Komagataella phaffii (Pichia pastoris), for example, is commonly used to manufacture protein therapeutics at large scales (~109 annual doses) in LMICs. We used K. phaffii to produce the SARS-CoV-2 spike protein receptor binding domain (RBD), a promising vaccine antigen found in many recent vaccine designs because of its small size and high density of neutralizing epitopes.

Here, we present a two-pronged approach of host-cell and protein engineering to improve global access to RBD-based vaccines. We genetically engineered K. phaffii to secrete the RBD without typical methanol-induction, which improved upstream titers ~5x and enabled longer production campaigns. We also rationally engineered an RBD protein with improved manufacturing titers and enhanced immunogenicity and antigenicity, enabling smaller dose sizes for wider vaccine distribution. The platform for RBD manufacturing presented here has been transferred to a GMP facility for manufacturing and clinical trials of engineering RBD antigens. This translational success suggests that the strain and protein engineering techniques presented here will be applicable to a wide range of protein therapeutics.

Tuesday
Strategies for control of mRNA-1273 and applications to an mRNA-LNP platform
03:20pm - 03:40pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Jack Kramarczyk, Presenter, Moderna, Inc.
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Moderna has established a platform for designing and scaling mRNA medicine, and mRNA-1273 has greatly accelerated the company's maturation. The rapid response to the global COVID-19 pandemic has been enabled by the implementation of foundational infrastructure and ten years of platform development. The manufacturing processes and control strategies are unique to the mRNA-LNP platform and have enabled a well-controlled and highly characterized pipeline of investigational vaccine candidates with industry-leading product profiles. In one example, a unique raw material control strategy protects product potency. In another example, a statistical model which estimates a mechanistic model has allowed high-confidence prediction of stability performance to enable 5C storage conditions for drug product. State-of-the-art technologies and control strategies established for mRNA-1273 are then applied across a pipeline of Moderna products.
Tuesday
Panel Discussion
03:40pm - 04:00pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Advances in Cell Culture Media and Novel Modalities: Advances in Cell Culture Media and Novel Modalities
04:30pm - 06:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Susan Sharfstein, Presider, SUNY Polytechnic Institute; Jeraldine Mendoza, Presider, ‍ ; Andrew Yongky, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

The growth of mammalian cells in chemically defined, nutrient-enhanced media is a cornerstone technology for basic and applied biomedical and biotechnology research. The formulation of culture media can have a substantial impact on cell behaviors, such as cell growth, viability and productivity. Medium formulation is profoundly intertwined with the cell’s metabolism and its response to the physicochemical environment. Manipulation and optimization of medium components have become increasingly specific and sophisticated in order to achieve the desired culture phenotypes, high intensity cell culture production, and/or certain product quality attributes. Such rational design will move engineering and discovery forward by supporting reproducible research across labs and by enabling more productive and better controlled cell culture systems. This session will focus on the interplay between media, growth conditions (e.g., pH, dissolved oxygen, and feeding strategies) and aspects of cell physiology including genotypes, phenotypes, and metabolic pathways. Papers relevant to these topics are highly encouraged, including but not limited to those focusing on cell culture medium optimization, medium chemistry understanding, medium impact on cell function and metabolism, effects of media in controlled environments and 'omics analyses of mammalian cells in varying culture conditions.

Tuesday
Circadian regulation of cytochromes P450 in 3D hepatocarcinoma spheroids
04:30pm - 04:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Andre Rodrigues, Presenter; Vibha Narayanan, Rensselaer Polytechnic Institute; Jonathan Dordick
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Circadian rhythms characterized by 24 h oscillations, also known as the circadian clock, is universally present in all organisms and regulate physiological functions based on the 24 h structure of a day. Circadian disruption has been implicated in the pathogenesis of aging and disease, including diabetes, cancer and neurodegenerative disorders such as Alzheimer’s Disease and Parkinson’s Disease. While the internal clock synchronizes gene expression with the guidance of external cues like light, a similar synchronization of gene expression can be induced in vitro by incubating the cells with an increased percentage of serum followed by rapid removal. This ability to mimic gene synchronization in vitro provides an opportunity to understand the influence of the circadian clock on expression of drug metabolizing enzymes, most specifically the cytochromes P450. Previous studies have suggested that synchronization of HepG2 cell line induces the rhythmic gene and protein expression of CYP3A4, CYP2D6 and CYP2E1. However, further insight on the role played by the 3D microenvironment on the rhythmicity of these genes and the corresponding enzymatic activity is lacking. To understand this interplay, gene expression of the circadian machinery and CYP450s were compared using the model human hepatocarcinoma cell line, HepG2. Upon cell synchronization by serum shock, gene and protein expression of core clock regulators and CYP450 enzyme activity were assessed using RT-PCR, westerns, and selective enzyme assays. Rhythmic expression of these genes was demonstrated. Further insight into the influence of the 3D microenvironment on circadian regulation of the CYP450s were then obtained. This work provides a better understanding of chronopharmacokinetic events in humans by using physiologically relevant 3D culture systems.

Tuesday
Effect of vitamins and metal ions on productivity and charge heterogeneity of IgG1 expressed in CHO cells
04:50pm - 05:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Neelesh Gangwar, Presenter; Rishabh Mishra; Niharika Budholiya; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Background: Recombinant monoclonal antibodies have emerged as the most successful modality of biotherapeutics. They are primarily expressed in Chinese Hamster Ovary (CHO) cells. It is well known that post-translational modifications (PTM) contribute significantly to heterogeneity with respect to charge, glycosylation, and size. These attributes in turn may impact stability, pharmacokinetics, and pharmacodynamics of the biotherapeutic product. Cell culture media components are known to significantly contribute to both cellular productivity as well as post-translational modifications. In recent times, chemically defined (CD) media are replacing complex undefined media so as to improve consistency in process performance and product quality. Thus, it is highly desirable to understand how media components affect product quality.
Method and Results: This study aims to explore the impact of vitamins and metal ions on protein expression and post-translational modifications specifically charge heterogeneity. Biotin, choline chloride, D-calcium pantothenate, folic acid, pyridoxine hydrochloride, thiamine hydrochloride vitamins and Fe, Cu, Mg, Co, Zn, Mn, Ni metal ions were examined in this study. The results indicate that pyridoxine enhances productivity while Zn, Cu, Fe, Mn, and biotin impact charge heterogeneity. While, Fe, Mn and Ni enhance production of the acidic variants Cu and biotin inhibit it. Zn reduces formation of basic variants while biotin enhances them.
Conclusion: The results from this investigation could be used for process control so as to get consistent charge variant profile, in particular for biosimilar products.

Tuesday
Evidence that lactate is oxidized in the mitochondria of proliferating cells
05:10pm - 05:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Xiangfeng Niu, Presenter, Washington University in St. Louis; Gary Patti
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Increasing evidence supports that proliferating cancer cells use lactate as a source of energy and a carbon source, however, determining whether lactate is oxidized in the cytosol or mitochondria is an experimental challenge. Here, we apply three independent strategies to confirm that lactate is oxidized in mitochondria: (i) isotope tracer analysis, (ii) transport-exclusion pharmacology, and (iii) genetic manipulation of putative proteins.
(i) Oxidation of 2-2H-lactate by lactate dehydrogenase (LDH) produces unlabeled pyruvate and 2H-NADH, which cannot directly cross the inner mitochondrial membrane. The metabolism of proliferating cells given 2-2H-lactate was quenched and their mitochondria isolated. Mass spectrometry analysis showed 2-2H-lactate in mitochondria, supporting direct import.
(ii) LDH has a binding site for pyruvate/lactate and a binding site for NADH/NAD+. The enzyme can therefore be inhibited by chemical mimetics of either pyruvate or NADH. We exploited a chemical mimetic of pyruvate able to cross the inner mitochondrial membrane and a chemical mimetic of NADH that could not. Interestingly, the inhibitors show different activities, supporting the presence of LDH in a compartment (e.g., mitochondria) that is inaccessible to a chemical mimetic of NADH.
(iii) We found that LDHB uniquely localized to mitochondria of proliferating cells. After knocking out LDHB, we found that isolated mitochondria from these same cells could no longer readily oxidize lactate.

Tuesday
Host miRNA biomarkers for viral infection in mammalian cell production cultures
05:30pm - 05:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Adventitious viral infection in mammalian cell production cultures is of great concern to the pharmaceutical industry, due to its far reaching economic and public health impacts, and the difficulty of detection. Current viral detection techniques can be costly, time consuming and labor intensive, and have a high risk of false positive or negative results. We propose a novel method for detecting viral infection of cell cultures through monitoring the host cell miRNA transcriptome as a real-time, direct measurement of cellular stress. This method would identify states of infection independent of the infecting virus, so that unknown viruses could also be detected. miRNA is an ideal biomarker for this application due to its homology across species, prevalence, accessibility, and role in maintaining cellular homeostasis. Here, miRNA transcriptome changes were measured in purposely-infected CHO-K1 cultures at two time points with four different viruses – Reo3, MMV, PI2, and EMC. It was found that miRNA expression changed significantly based on both culture age and viral infection status. Seven miRNAs showed significant fold change in more than one type of viral infection on day 3 post-infection, including cgr-miR-21-3p, cgr-miR-1260, cgr-miR-3068, and cgr-miR-30d. Many more miRNAs showed significant fold change on day 7 post-infection for multiple types of viral infection, most notably, cgr-miR-210 and cgr-miR-222-3p. In conclusion, these potential biomarkers for general viral infection in mammalian production cultures lay the groundwork for development of a quick viral detection method that can be implemented at multiple points in upstream bioprocessing.
Tuesday
Gene therapy for neurological diseases
05:50pm - 06:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 03
Junghae Suh, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Decades of biological research have unveiled the genetic cause of many debilitating diseases that affect the central nervous system (CNS). We are at a point where we aim to translate that knowledge into potential one-time treatment options and curative therapies. Gene therapy as a therapeutic modality is continuing to evolve and we have seen important advances propelling the field forward. However, additional focused innovation is needed to overcome the complex challenges in gene therapy for neurological diseases. Challenges regarding the delivery platform, cargo, vector production, and vector performance characterization as well as potential solutions to these challenges will be discussed.
Downstream Processing: in Silico & Mechanistic Modeling of Downstream Bioseparations: In Silico & Mechanistic Modeling of Downstream Bioseparations
04:30pm - 06:30pm 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
In silico framework for multi-scale technology evaluation in monoclonal antibody (mAb) manufacturing
04:30pm - 04:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Processes for monoclonal antibody (mAb) production are often directly scaled up from lab scale to commercial scale to reduce time-to-market, and therefore, more optimal process alternatives are neglected. However, growing economic constraints are driving the development of new processes, which requires a systematic and quantitative evaluation of the design space. Because of the synergistic effects of product-process decisions, it is important to account for process alternatives such as novel flowsheets or new technologies already in the product design phase, hence solving the integrated product-process design problem.

The design problem for mAb manufacturing encompasses knowledge in multiple scales: from mechanistic understanding and experimentation, to unit operation, to process flowsheet. Methods and tools need to be developed to build understanding and evaluate alternatives across all scales.

Let us consider the evaluation of new technologies and/or approaches for mAb manufacturing that have been developed, including alternative expression systems, non-chromatographic separations, or continuous manufacturing. A flowsheet model represents the production process, including interactions between process steps, which can be used to test ideas for improvement as well as to identify process steps as focus for development. Moreover, flowsheet models allow the evaluation of performance criteria that drives decision making, such as capacity, economics and sustainability. Unit operation models are then used to, at a higher level of resolution, identify values of key variables and parameters to achieve the targets set by testing at the flowsheet level. This is then used to drive experimentation by defining materials and methods, e.g. experimental conditions to be tested. This flow of information across scales applies in both directions.

The objective of the presentation is to present an in silico, systematic framework for multi-scale process design and technology evaluation for biopharmaceuticals. The method will be exemplified through a case study for a mAb process starting with the development of a discrete event simulation-based flowsheet model and its application to evaluate and screen ideas for increasing capacity. This leads to the selection of a new technology for upconcentration and drives CFD modelling at the unit operation level, which then drives model-based experimental identification.

Tuesday
Role of mechanistic modeling in bioprocess control
04:50pm - 05:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Dr. Anurag S Rathore, Presenter, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The initiatives of Quality by Design (QbD) and Process Analytical Technology (PAT) have gained momentum in the biopharmaceutical industry. This has also renewed interest in mechanistic modelling of biotech unit operations and using these model for process optimization and control. This talk will highlight the benefits that the industry can gain by performing mechanistic modelling. Two case studies will be presented, targeting the two major unit operations in bioseparations, process chromatography and ultrafiltration. In the first case study prediction of the impact of variability in feed quality and in gradient shape on separation of charge variants by cation exchange process chromatography has been attempted to facilitate feed forward control. Five batches having different compositions of charge variants have been used to demonstrate the proposed pooling strategy based on simulated chromatograms and the outcome has been compared to offline pooling based on fractionation. For all the conditions examined and for the desired target of main product (67%), the proposed approach resulted in remarkable consistency in product quality (67±2%) while delivering a yield of greater than 90%. In the second case study, ultrafiltration of a monoclonal antibody product to a high concentration (> 150 mg/ml) has been modelled. It has been seen that when protein concentration goes to such high level, deviations in pH and excipient concentrations are observed. Mechanistic model of the step has been created and used to improve process control. The two case studies showcase the utility of process modelling in process optimization and control.
Tuesday
Model-based chromatography process development for complex vaccine mixtures
05:10pm - 05:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In recent years, the biopharmaceutical industry recognized the added value of mechanistic modeling techniques as they provide an extended level of process understanding. Mechanistic models, once calibrated, enable to run numerous virtual experiments in an inexpensive and safe way, thereby reducing time and material consumption. However, chromatographic mechanistic models rely on adsorption equilibrium isotherms of the product and other impurities towards the resin. The experimental determination of model parameters can be a tedious task and could partially be automatized using High Throughput Experimentation techniques.
It becomes even more complicated for complex mixtures containing numerous unknown components. The same is applicable for vaccines upstream feeds containing thousands of host cell proteins (HCPs) and other cell related impurities, especially at the capture step which is performed in the presence of large amounts of impurities and aims to achieve an initial quite high product purity by removing most of these impurities. Determination and modeling each components adsorption isotherm would require an unreasonable effort in both time and costs, hence it is unnecessary to model each component of the mixture individually. This presentation will show a chromatographic modeling approach to simulate complex vaccines mixtures by making use of pseudo-component adsorption isotherms for HCPs.

Tuesday
Modeling, design and performance evaluation of high capacity and high productivity novel chromatography membrane materials with non-spherical diffusive structures
05:30pm - 05:50pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
In chromatographic bind and elute processes of proteins like e.g. mAbs, spherical particles of 50 to 100 µm are most applied. Due to these large diffusive distances, particle-based processes are limited by diffusive mass transport. In contrary, purely convective membranes or monoliths show no diffusive mass transfer limitations but exhibit a strong trade-off between permeability and binding capacity, leading to pressure-drop and scalability issues.
To overcome these limitations, alternative chromatographic membranes are being developed, combining structural and performance aspects of particles and purely convective materials. These novel membranes provide chromatography phases that are highly accessible by diffusion and at the same time have high binding capacities. By that, they overcome mass transfer, pressure-drop and scalability limitations of above-mentioned materials while maintaining a high binding capacity. However, designing these materials by mechanistic modeling is conceptually not possible with common chromatography models because these models assume spherical diffusive phases, whereas in the novel membranes the diffusive phase exhibits convex, concave, and plane surfaces.
In this work, an approach is presented which enables the mechanistic modeling of mass transfer in alternative chromatography materials whose diffusive phase geometry differs from spheres. The geometry of the diffusive phase of experimentally derived samples of these novel membranes was analyzed by 3-D scans using a confocal laser scanning microscope. These geometric features were used to model the novel membranes with CADET. The chromatographic models available in CADET were extended to describe plate and cylindrical geometries.
With this extension, we were able to predict the concentration profile inside the novel membranes, the breakthrough curves and the overall chromatographic performance. Based on the microscopic analysis of Protein-A affinity prototypes of these novel membranes, breakthrough curves were successfully predicted. Furthermore, by optimizing design parameters e.g. the diffusive length, the productivity of the chromatographic process can be increased by more than an order of magnitude compared to state-of-the-art particles while maintaining the binding capacity in the range reached by those particles. Experimentally validated results will be presented to underline the potential of the novel membranes, combining high productivities with high binding capacities.

Tuesday
Mechanistic modeling of multimodal chromatography for separation of a monoclonal antibody from product-related impurities: Fab fragment and aggregates
05:50pm - 06:10pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The specific selectivities offered by multimodal ligands drive the increased application of multimodal chromatography in the purification of complex new ”multispecific” antibodies. Together with stronger requirements to remove product-related impurities and robust purification processes, this requires improved understanding of the protein-multimodal ligand interaction mechanism. The goal of the present study is to develop a mechanistic model to predict monoclonal antibody (mAb)-Fab fragment and heterogenous aggregates separation based on the preferential interaction adsorption model developed by Perkin et al. (1997). The fit of the adsorption isotherm to isocratic elutions of mAb, Fab fragment, and aggregates result in a characteristic ‘U’-shaped curve for mAb, Fab, and aggregates retention factors as a function of NaCl salt concentrations. The ‘U’-shaped curve of retention factor is attributed to the mixed interactions of the electrostatic interactions at low salt concentration and the hydrophobic effects at high salt concentration. The retention behavior of mAb, Fab, and aggregates under linear and step gradients were used to estimate the preferential interaction model parameters of each on Capto™ MMC ImpRes multimodal column using isocratic fit parameters as the initial guess value. The proposed mechanistic model successfully simulates the elution peaks for both the regimes of low and high salt concentrations, and can therefore be used for purification process optimization and control for separation of process-related impurities.
Tuesday
A first-principles affinity complex titration isotherm for protein A chromatography
06:10pm - 06:30pm USA / Canada - Eastern - August 24, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Although the Langmuir isotherm is frequently used for mechanistic modelling in Protein A (ProA) chromatography, it must be empirically manipulated to capture the relationship between pH and monoclonal antibody (mAb) retention during loading, washing and elution. And while empirical isotherm expressions incorporating the pH-dependence of retention have been developed by others, they cannot provide mechanistic insight into chromatographic behavior. We have considered the titratable residues within the primary mAb-ProA binding site as well as the maximum mAb-ProA binding stoichiometry within a given resin to propose a new, fundamental isotherm. We have demonstrated the model’s practicability and feasibility via experiment and simulation for a broad range of breakthrough loading and pH step elution and gradient elution schemes with a model mAb and MabSelect SuRe media. Besides the isotherm parameters, the main input to the chromatographic simulations is the pH profile used. The correlation between gradient experiments and simulations is good, supporting routine industrial and academic application. Further, we demonstrate that the chromatogram for a complete loading, washing and elution cycle can be accurately described with a single set of parameters. The isotherm also provides a framework for parsing mass transport details from binding details in the analysis of chromatograms and provides mechanistic insight into the energetics of binding as a function of pH.