Biomedical Engineering and Bioengineering Commons^™

A Novel Technique To Characterize The Surface Hydrophobicity Of Proteins Using Inverse Liquid Chromatography, Dilip Sethi, Sarah Hedberg, Daryl Williams

Biological and Pharmaceutical Complex Fluids III: Protein Self-Assembly, Rheology and Interfacial Properties

This work presents a novel technique to characterize the surface hydrophobicity and other surface properties of proteins. The surface properties of industrial and therapeutic proteins are key to understanding their behavior in-situ, in the laboratory and in processes. Protein surface hydrophobicity is a marker for three dimensional structure, stability and function. Inverse Liquid Chromatography of Proteins (ILCP) with small molecule hydrophobic probes can be used to obtain direct measurements of the surface hydrophobicity of column resin-bound proteins using changes in probe retention behavior. The dimensionless hydrophobicity factor (H_f) for Lysozyme and BSA was obtained with changing pH …

Biophysical Characterization Approaches To Aid The Selection Of Protein Formulations By Predicting Their Physical Stability During Long-Term Storage, Hristo Svilenov, Gerhard Winter

Biological and Pharmaceutical Complex Fluids III: Protein Self-Assembly, Rheology and Interfacial Properties

The formulation of therapeutic proteins is a critical process which aims at finding the most suitable conditions that impede protein degradation during long-term storage. One degradation path of high interest is the non-native aggregation¹. The latter can be greatly suppressed by the selection of suitable solution conditions². Over the years, various biophysical techniques have been explored as tools to quickly select the most promising formulations for long-term storage.

In this talk, we share the experience in our lab how some of these techniques can be integrated into protein formulation studies. We discuss the application of differential …

Aggregation Challenges In The Formulation Development Of Multi-Dose Peptide Products, Jingtao Zhang, Katelyn Smith, Wei Xu, Yongchao Su, Suzanne D’Addio, Yogita Krishnamachari, Jameson Bothe, Daniel Yin, Xinpei Mao

Biological and Pharmaceutical Complex Fluids III: Protein Self-Assembly, Rheology and Interfacial Properties

The formulation development of parenteral peptide therapeutics frequently encounters aggregation challenges. In-depth biophysical understanding of the molecule and formulation are required to achieve formulation robustness. Further, unique considerations need to be given for peptide products that require multi-dose as the use of preservatives can promote aggregation while preservative effectiveness can also be impacted by its interaction with the peptide. This presentation will focus on the reversible and irreversible fibril aggregates in peptide formulations. Biophysical characterization of aggregation and formulation will be discussed in detail. Formation of reversible aggregates and the impact of excipients especially preservatives will be discussed. For the …

Conference Program, Samiul Amin, Paolo Arosio, Miguel Rodrigues

Biological and Pharmaceutical Complex Fluids III: Protein Self-Assembly, Rheology and Interfacial Properties

No abstract provided.

Conference Program, Erin Buckley, Christophe Moser, Brian Pogue, David Sampson

Advances in Optics for Biotechnology, Medicine and Surgery XVI [2019]

No abstract provided.

Novel Supply Chain And Process Modeling For Cell Therapy Manufacturing And Distribution, Chip White, Ben Wang, Kan Wang, Reid Bishop, Aaron Levine, Brian Liu, Bhavya Divvela, Aubrey Incorvaia, Kris Saha, Tava Das

Advancing Manufacture of Cell and Gene Therapies VI [2019]

We present a two-level hierarchical supply chain model of (autologous) CAR-T cell therapy that serves as the basis for the development of strategies to: 1) deliver cell therapy products that are safe and have a high level of efficacy, 2) minimize fulfillment time and variability, and 3) reduce total manufacturing and logistics costs while reducing the risk of patient morbidity and mortality. The model consists of two integral components: (1) an agent-based program for a “single manufacturing facility” that simulates the manufacturing and quality control process of cell therapy; and (2) a supply chain network program that evaluates different supply …

Key Engineering Challenges In The Biomanufacturing Of Lentiviral Viral Vectors, Peter Jones

Advancing Manufacture of Cell and Gene Therapies VI [2019]

No Abstract.

Tangential Flow Filtration And Scalability In Viral Vector Purification, Eni Sterjanaj, Heather Mall, Rachel Legmann, Jacky Dang

Advancing Manufacture of Cell and Gene Therapies VI [2019]

Pall Biotech has linearly scalable tangential flow filtration technology that can cover processing volume ranges from less than a liter to over 2000 liters. Manual assemblies can be used with Pall TFF filters in scales under 20L and programmable skids are available for larger scales. In this document TFF linear scalability of up to 200 liters is covered starting from a 7L initial volume and maintaining similar pressures, processing times and yields at all scales. Using this scalable technology Pall biotech can help biologics companies go to clinical trials and to commercial scale manufacturing successfully.

Please click Additional Files below …

Conference Program, Dolores Baksh, Rod Rietze, Ivan Wall

Advancing Manufacture of Cell and Gene Therapies VI [2019]

No abstract provided.

Workshop Agenda, Fernanda Masri

Advancing Manufacture of Cell and Gene Therapies VI [2019]

No abstract provided.

Development Of A Closed Car-T Manufacturing Process, Loo-Yong Steven Kee, Calley Hirsch, Devina Ramsaroop, Elizabeth Csaszar, Danylo Sirskyj, Aaron Dulgar-Tulloch

Advancing Manufacture of Cell and Gene Therapies VI [2019]

The field of immunotherapy has emerged as a promising new type of treatment for cancer with the approval of the first two CAR-T therapies. The clinical success of T-cell based immunotherapies necessitates a robust manufacturing process for these products to be consistently produced at commercial scale. Our CAR-T workflow combines unit operation specific solutions for thaw of an apheresis unit, wash, CD3 selection, T-cell activation, lentiviral transduction, incubator- and reactor-based expansion culture, harvest, formulation, cryopreservation and thaw of CAR-T product. We have evaluated the impact of both serum-containing and xeno-free culture media, commercially available T-cell selection and activation reagents, closed …

Scale-Up Study For Ex-Vivo Expansion Of Allogeneic Natural Killer Cells In Stirred-Tank Bioreactor, Juyoung Kim, Hyung Jin Nam, Sang Hyun Lee, Jong Beom Ku, Seung Ryel Han, Hye Jin Shin, Yu Kyeong Hwang, Sang Hoon Paik

Advancing Manufacture of Cell and Gene Therapies VI [2019]

Natural killer (NK) cells are a type of lymphocyte in the blood that are responsible for innate and adaptive immune response, and they mature in the liver and bone marrow. Being a key role in host defense system with direct and indirect killing of virus-infected cells or cancer cells, NK cell has been considered an attractive candidate for cancer therapy. Peripheral blood shows the low frequency of NK cells, so ex vivo expansion method is important to obtain sufficient NK cells for therapeutic use. Currently, we successfully developed bioreactor process for NK cell expansion on lab-scale. Stirred-tank bioreactor could be …

Single Use Disposable Biosettler Removes The Dead Cells And Cell Debris Selectively To Increase The Viability Percentage Of Mammalian Cells (E.G., Car-T) During Expansion, Dhinakar Kompala

Advancing Manufacture of Cell and Gene Therapies VI [2019]

Current challenge in a FDA approved cell therapy product for adult B-cell leukemia is reported to be the percentage of viable cells after manufacturing is sometimes out of specified range. As the head of a large contract development and manufacturing organization observed, this fall in viability percentage during CAR-T cell manufacturing is a challenge to the whole industry.

We present a simple and powerful off-the-shelf solution to this big challenge in all mammalian cell culture expansion bioreactor system. Our single use disposable BioSettler has been demonstrated to be uniquely capable of removing dead cells and cell debris selectively from the …

Fed-Batch Process Development Using Metabolically Efficient Cho Cells, Cecile Toussaint

Cell Culture Engineering XV

The development and application of fed-batch strategies have greatly contributed to the improvement of the productivity of cell culture processes. However, in such processes, lactate and ammonia accumulation in the culture medium over time can have detrimental impacts on cell growth and product quality. Continuous cell lines typically exhibit an inefficient metabolism whereby most of the pyruvate derived from glucose is diverted to lactate and only a small percentage is incorporated into the TCA cycle. Thus, further process improvements can be expected by combining the application of rational fed-batch strategies with targeted metabolic engineering of cells to reduce waste metabolite …

Deepening Knowledge On Cho Cells Metabolism Using Multiple Tracer Substrates, Ana Teixeira, Manuel Carrondo

Cell Culture Engineering XV

Optimization of culture conditions through trial-and-error has led to much of the past progress on animal cell culture. Today, a renewed account of the underlying biologies through a “systems” view is providing a deeper knowledge of cellular physiology and laying the basis to reengineer desirable states of further increased culture performance. In this work, we took a comprehensive view of cellular metabolism by extensively profiling extracellular and intracellular metabolomes after ¹³C‑label supplementation in parallel labelling cultures of [1,2-¹³C]Glucose, [U-¹³C,¹⁵N]Asn, [U-¹³C,¹⁵N]Ser and [1-¹³C]Pyruvate. This integrative approach was used …

A Holistic Approach To Facility Protection From Adventitious Agents – A Case Study, Matthew Osborne, Ronan Kelly, Ann Maria Mccrohan, Marie Murphy

Cell Culture Engineering XV

The Eli Lilly biologics manufacturing facility in Kinsale, Ireland has been operational since 2010 with a 100% cell culture contamination control success rate. The presentation will review the holistic approach to facility protection from adventitious agents that underpins this success including:

• The risk assessment approach to points of entry and management via detectability and control measures
• The approach to personnel training that considers human factors, increased vigilance and event simulations following strategies that are used in chemical synthesis process safety

The presentation will then focus on control of adventitious virus. The talk will briefly comment on the early warning measures …

Nanofiltration As An Effective Means To Prevent Virus Contamination Of Cell Culture Processes, Christina Carbrello

Cell Culture Engineering XV

While cell culture media is typically sterile filtered to remove bacteria and mycoplasma, many bioreactors remain unprotected from viral contamination. As awareness of the risk for viral contamination of cell culture processes grows, manufacturers are seeking virus barrier technologies to remove or inactivate viruses. Filtration is an ideal technology in terms of familiarity, ease of use and scalability. However, hurdles to its implementation remain, most notably, the high cost and large footprints required to implement filters designed for the downstream process and the concern that the small pore size of virus filters could impact cell culture performance. A filter specifically …

Development Of An Enriched Cho Feed Media For Quality Therapeutic Antibody Production From High Performing Clones, David Ho

Cell Culture Engineering XV

The use of feed media during protein production is essential to sustain viable cell densities and facilitate high productivity for modern high performing clones. However, the introduction of feed media can also have a significant impact on product quality attributes, such as glycosylation for therapeutic antibodies, which can ultimately impact therapeutic efficacy. For certain antibodies, the absence of fucose on the N-glycan at Asparagine 297 of the Fc domain has been shown to significantly increase binding to the FcgammaRIII receptor on NK cells and results in increased ADCC activity. Here we have incorporated product quality analysis including N-glycan analysis into …

Using Definitive Screening Design To Effectively Assess The Combinatorial Impacts Of Media Supplements On Monoclonal Antibody Production In Mammalian Cells, Aaron Chen

Cell Culture Engineering XV

Process understanding is an essential component of QbD principles. Cell culture media composition can impact many process performance parameters such as viable cell density, product yield and product quality attributes. In order to enhance process understanding and exert better process control, numerous media supplements have previously been tested. However, their combinatorial effects on process and product quality have largely been unexplored. This is partially due to the resource and efforts required to execute relatively large size of experiment and data analysis.

Recent advancement in modern design of experiment methodology has alleviated this issue. The definitive screening design (DSD) allows for …

Adapting An In-Licensed/Acquired Cell Culture Process To Platform Conditions, Raghunath Shivappa

Cell Culture Engineering XV

The use of platform materials and processes for new products help streamline process development, enhance predictability and efficiency during manufacturing, and simplify the raw materials supply chain. Although common, adaptation of cell expression systems can require a significant amount of process development work to achieve desired product yield, quality, and comparability. Here, we present a progressive development approach for evaluating platform media and feeds for an acquired cell culture process.

A CHO-DG 44 cell line expressing a novel antibody product was procured through acquisition. The original non-platform media and fed-batch process yielded titers of approximately 3 g/L. A study was …

Antibody Production With Site-Specific Non-Natural Amino Acid Incorporation For Generation Of Antibody Drug Conjugates, Alyssa Powell

Cell Culture Engineering XV

Ambrx’s mammalian expression platform (EuCODE) enables site-specific incorporation of non-natural amino acids into antibodies. This EuCODE technology provides a means for stable payload linkages at defined sites and with a defined DAR of 2 for antibody drug conjugate (ADC) generation. While the ability to control the DAR and payload site can provide an advantage to an ADC, the incorporation of the non-natural amino acid into the antibody heavy chain introduces a unique challenge for antibody production.

To achieve high production of monoclonal antibodies (mAbs) containing non-natural amino acids, we engineered a CHO-K1 cell line which stably contains Ambrx’s patented tRNA …

Evaluating Sugar-Based Detergents As A Potential Alternative To Poloxamer Bubble Protectant, Jessica Wuu, Jayanthi Lakkyreddy, Steven Meier

Cell Culture Engineering XV

Poloxamer 188 (commonly referred to as Pluronic® F-68) is a commonly-used medium component which protects against bubble-induced shear in mammalian cell cultures. Recently, concerns have arisen due to limited sourcing and variable performance of this critical component. In 2008, Hu, et al.¹ proposed the use of sugar-based detergents – specifically, maltopyranosides and glucopyranosides – as potential bubble protectants based on their rheological properties and relatively low toxicity to cells. This work explores the feasibility of these detergents using commercially relevant CHO cell lines and media. Detergent candidates were evaluated based on their tolerance by cells, stability in cell culture …

Adaptation Of Cho Metabolism To Long Term Phosphate Limitation, Mugdha Gadgil

Cell Culture Engineering XV

Phosphate is an important component of abundant intracellular molecules like RNA, is a critical component of energy metabolism and is involved in substrate phosphorylation in cellular metabolism and signaling. Inorganic phosphate (P_i) is transported through specialized transporter proteins and limitation in the availability of phosphate in the growth medium limits cell growth. P_i concentrations have been reported to regulate the rates of aerobic glycolysis and oxygen uptake. Thus it is expected that cellular metabolism will adapt to long term phosphate restriction. Since complete phosphate deprivation will result in lack of growth, such an adaptation needs to be …

Impact Of Poloxamer 188 Variability On Biologics Manufacturing: Mitigations And Causal Investigation, Salim Charaniya

Cell Culture Engineering XV

Poloxamer 188 (P188), a water-soluble, synthetic copolymer with hydrophilic as well as lipophilic properties, is widely used as a cell protectant for bubble-induced shear during cultivation of suspension-adapted mammalian cells for large-scale production of protein-based therapeutics. Poor cell growth and lower product yield has been recently observed at commercial scales in more than one Chinese hamster ovary-based manufacturing process within Roche network when specific lots of P188 were used. An investigation is being conducted to unearth the root cause of “poor-performing” P188 lots and to identify means to mitigate the product supply risks. P188 raw material is currently sourced from …

A Host Cell Protein That May Impact Polysorbate Degradataion, Kelvin Lee

Cell Culture Engineering XV

There is growing interest and concern related to host cell proteins. Current issues with host cell proteins include questions related to the various available analytical approaches and the sensitivity of those approaches as well as issues with purification strategies and the impact of host cell proteins downstream. The availability of a sequenced genome enables new opportunities to address problematic host cell proteins via cell line engineering. We studied difficult to remove host cell proteins in CHO cells with the goal of identifying host cell proteins that may be product associated with a number of monoclonal antibodies, that may copurify with …

Development Of Antibody Detection Methods For Active Product At The Cell Culture Stage, Gregory Walsh, Matthew Schraut, Jon Barricelli, Lada Laenen

Cell Culture Engineering XV

Development of new analytical methods to characterize the manufacturing process became necessary when the site wanted to implement process improvements to increase output from the bioreactor. The bioreactor process is monitored and controlled for dissolved oxygen content, glucose concentration, temperature, etc. and samples of the harvest are analyzed daily using an endpoint assay and a total protein method. Neither of the two methods can detect the total amount of product at the harvest stage; the endpoint assay will only detect active product and the total protein method will not discriminate between the cellular proteins and the active product. We have …

Development And Application Of Glycosyltransferases For In Vitro Glycoengineering, Alfred Engel

Cell Culture Engineering XV

Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity, e.g. IgG featuring terminal sialic acids were shown to suppress inflammatory response (ADCC) and increase serum half-life. Fully galactosylated structures seem to improve binding to the complement system (CDC).

Despite all efforts in cell-line engineering and process optimization to increase glycosylation homogeneity, today therapeutic proteins still show a heterogeneous glycosylation pattern, with large variations between bioprocesses and even from batch to batch. The use of glycosyltransferases for enzymatic synthesis of well-defined glycan structures will become an essential tool – at least in …

Comparison Of Commercial Cho Cell Media Formulations Using Material-Oriented Recurrent Spectral Libraries, Kelly Telu

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are commonly used for the production of biological therapeutics. Metabolic profiles of media components can be used to monitor process variability and look for markers that discriminate between batches of product. Currently, there exists no database of CHO media components or a method for the comparison of commercial media formulations. We are creating material-oriented libraries of CHO media components that include LC-MS/MS and GC-MS data. The libraries represent all metabolites that can be detected by LC-MS/MS and GC-MS and consist of recurrent spectra that cover all known fragmentation conditions and precursors. Recurrent spectra occur repeatedly …

Integrating Emerging Trends In Upstream Process Development: Autosampling, Nutrient Feedback Control, And Single Use Tanks, T. Craig Seamans

Cell Culture Engineering XV

Key to effective upstream development is the presence of resource-efficient systems for examining complex process options. In this presentation, we report the design and characterization of a 3-L rigid single-use bioreactor (SUB), combined with a completely automated sampling and control system capable of evaluating different metabolic-based feed strategies.

Considerations in our development of a custom 3-L SUB, targeted to replace current workhorse glass reactors, encompassed proper technical functionality to flexibility and ease of use. Highlights include: vessel configuration details (impeller, sparger, baffles), perfusion readiness, agitation system design (magnetic bottom drive), and extra sensor ports and feed lines. The SUB was …

Incidence And Potential Implications Of Methylglyoxal In Industrial Cell Culture Revisited, Frank Chaplen

Cell Culture Engineering XV

Methylglyoxal is a toxic by-product of glycolysis and amino acid metabolism in mammalian systems. The major route for methylglyoxal detoxification is the glyoxalase pathway, which consists of the enzymes glyoxalase I (GLO1) and glyoxalase II (GLO2). A required co-factor for the glyoxalase pathway is reduced glutathione. Evidence suggests that GLOI and methylglyoxal have important roles to play in the signal pathways associated with oxidative stress and necrotic cell death¹. Previous work has demonstrated that growth conditions found in industrial cell culture have marked effects on endogenous methylglyoxal levels in Chinese hamster ovary (CHO)². Furthermore, decreased levels …