Engineering Commons^™

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 …

Anti-Oxidant Addition To Cd-Cho Media To Prevent Damage Induced By Uv Disinfection, Emma Dare

Cell Culture Engineering XV

Ultraviolet (UV) irradiation is an advantageous sterilization technique in the biopharmaceutical industry as it is capable of targeting non-enveloped viruses and smaller viruses that are difficult to remove via conventional filtration. Unfortunately, cell culture media contains many components that, upon exposure to light, undergo phototransformations that subsequently lead to poor cell growth. We have shown previously Chinese Hamster Ovary (CHO) cell culture performance was affected at fluences above 200 mJ/cm² due to the depletion of several key nutritional components (pyruvate, tryptophan, tyrosine, pyridoxine, pyridoxamine, thiamine) and the accumulation of reaction by-products (acetate, formate, sarcosine). Our objective was to determine …

Accelerated Bioprocess Characterization By Data Enrichment In Scale-Down Models, Viktor Konakovsky, Graham Mccreath, Jarka Glassey

Cell Culture Engineering XV

Scale-down models are often used for the definition of operating ranges in process development and validation and therefore data exploitation is an important task. Usually, the experimental scientists have very tight timelines and consequentially may often only perform the required routine of cleaning and sorting the data without more sophisticated analyses, even though it might improve data quality. This problem is more exacerbated by the rise of fully automated, miniaturized high-throughput equipment in up- and downstream process development where data processing automation is not just optional but a must. However, cleaned, sorted and time-aligned data alone do not guarantee a …

Conference Program, Kathy Chan

Cell Culture Engineering XV

No abstract provided.

Nmr-Based Design Of Chemically-Defined Protein-Free Feed Medium For The Cho Expression System, Marina Goldfeld

Cell Culture Engineering XV

Recombinant expression of antibodies and other biotherapeutics relies heavily on the use of CHO cell lines. The product expression levels in these cell lines are dependent on the feed medium, one of the most important components of the cell culture process. We have developed a systematic approach to designing a chemically-defined feed medium through collaboration between Merck’s Process Development and Assay Development groups. Historically, feed media have been derived from non-chemically defined sources, such as bovine serum and yeast hydrolysate. While these media support robust cell growth and productivity, their chemically-complex nature has hindered efforts to increase the cells’ productivity. …

Troubleshooting The Recover Of Mater Cell Bank For A Commerical Product, Mei Shao, Kishan Rao

Cell Culture Engineering XV

Poor recoverability was observed for the Master Cell Bank (MCB) during the preparation of Working Cell Bank for a commercial product. The presentation will describe the investigation process, root causes identified and corrective actions implemented. A new Working Cell Bank was successfully manufactured after implementing the corrective actions. Our strategy to test the WCB prior to commercial manufacturing will also be discussed. In addition, preventative actions were also implemented as a result of the event.

The Oxygen Binding Protein, Hemoxcell(R), Increases Cho Cell Growth And Extends Viability By Enhancing Oxygen Delivery, Katrin Braasch

Cell Culture Engineering XV

The continuous monitoring of cell growth and viability is an integral part of biopharmaceutical production. Measurements of changes in the polarizability of individual cells can identify early emerging sub-populations of apoptotic cells in a dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the trajectory of individual cells was tracked as they passed through a bank of electrodes (sensitivity: 0.1 µm; rate: 5 cells per second) designed to differentially perturb the cells according to their polarizability. This perturbation was recorded as a force index (FI), which was related to the electrical displacement of the cells. Using this …

Moving From A Bioreactor Scale-Up/Scale-Down Approach To A More Holistic Operational Design Space View, Gene Schaefer, Rick Yeager, Ted Deloggio

Cell Culture Engineering XV

Scale-up of bioreactor processes for biologic products has been an area of focus in the industry for over 50 years and the results have generally been successful through the application of a number of specific correlations across scales for mass transfer, mixing, etc. However, the ability to visualize and understand the bioreactor design space in terms of these parameters has generally been limited as has any general understanding of how close a given process comes to certain sensitivity limits at various scales. As part of a project which involved establishing scale-down models at two different sites with long-term objectives of …

Implementation Activities For A Chemically-Defined Media Platform To Minimize Media Variability Impact To Cell Culture Performance And Product Quality, Martin Gawlitzek, Brian Wong, Robert Shawley, Masaru Shiratori

Cell Culture Engineering XV

Chemically-defined media have been developed for CHO cell culture platform processes. During implementation of the media at multiple scales and sites globally, additional activities were required to seamlessly balance practical considerations with desired process and product quality performance. Three main focus areas were further investigated and optimized to enable the platform implementation: 1) media preparation, filtration, and post-filtration stability; 2) media and supplement viral barrier compatibility; and 3) and raw material variability considerations. Overall, the effort provided an outlined approach for media implementation in the global network including scale and site-specific case studies where novel options were required.

The data …

Challenges In The Development And Adaptation Of Platform Process To Existing Pipeline, Edmund Scarfo

Cell Culture Engineering XV

The development and use of platform processes have been adopted as an industry-wide strategy for enabling faster timelines for early clinical stages. However due to increasing product demands and the competitive landscape of antibody therapeutics in the industry, the development and use of a highly productive and economic upstream platform process has also become an adopted strategy. In keeping with industry standards, Takeda’s platform has evolved from upstream processes capable of achieving protein titers from 0.7-1.2 g/L to highly productive processes achieving titers in the range of 4.0-7.0 g/L. Unlike these earlier, low titer processes for the same molecules, the …

How To Select The Most Suitable Media For Your Cells, Marcella Dalm

Cell Culture Engineering XV

Many different media are available to culture CHO cells. Most are either growth supporting or productivity supporting. Ideally, the basal medium is a growth supporting medium, while the feed medium is supporting both growth and productivity. In this study two clones producing the same mAb are compared on their response to a range of basal media. Clone I is fast-growing and low-producing, while Clone II is slower-growing, but high-producing. A panel of eleven different basal media from different vendors was evaluated for growth, titer and metabolism.

An overview is given on the response of the cells to the different media. …

Evaluation Of Performance Enhancing Effects Of Supplementation With Complex Feed System And Supplements With Sheff-Cho Cd Complete Media In Cho-K1 And Cho Dg44 Cultures, John Menton, Kyle Liu, Sagar Kokal

Cell Culture Engineering XV

Biopharmaceutical companies routinely utilize feed systems for maximizing the yield of recombinant proteins in Chinese Hamster Ovary (CHO) cells. Synergies between plant-derived hydrolysates, yeast extracts and recombinant proteins have previously been demonstrated to enhance productivity in CHO fed-batch cultures. Based on this knowledge, Kerry has developed CHO specific ACF (animal component free) feed systems and supplements for improving the overall culture health and enhancing recombinant protein yields. The availability of protein-free (PF) versions of the feeds and supplements provides an option for production platforms with PF requirements. Kerry has developed Sheff-CHO CD complete a CD media designed to work synergistically …

An Addition Of Lithium Chloride Improves The Transient Gene Expression Yield In Cho Cells, Che Kim, Tae Kwang, Gyun Lee

Cell Culture Engineering XV

As hundreds of new recombinant therapeutic proteins enter into development each year, a cost-effective transient gene expression (TGE) system is required to assess these proteins at an early phase as a more rapid alternative to a stable gene expression system. While several strategies, such as genetic manipulation and environmental modification, have been attempted in TGE systems to improve the overall yield in Chinese hamster ovary (CHO) cells, chemical supplementation is often employed in industrial processes owing to its simplicity and effectiveness. Lithium chloride (LiCl), which is known to induce G2/M-phase cell cycle arrest, was found to be an attractive chemical …

Retrospective Implementation Of Quality By Design For Legacy Commercialized Enzyme Replacement Therapies, Anup Agarwal, Eric Hayduk

Cell Culture Engineering XV

Quality by Design (QbD) has been widely adopted by the pharmaceutical industry as a tool for transforming development, manufacture, and commercialization of drug products. QbD ensures that quality is built into a manufacturing process to consistently produce desired product. As per a FDA guidance¹, QbD should be employed at the product development stage to ensure manufacture of a product with predefined quality. Here we present a retrospective QbD approach employed at Shire to define an improved control strategy for a commercial process using risk-based process understanding and characterization. Key challenges and considerations of implementing a QbD strategy on …

Scaling Microcarrier-Based Cell Expansion Processes, Mark Szczypka

Cell Culture Engineering XV

Therapies utilizing stem cells isolated from various tissues are currently undergoing clinical evaluation. It is predicted that for many indications large quantities of cells will be required to treat the numbers of patients in need of therapy. This requirement represents a challenge because the surface area needed to generate the projected numbers of cells is significant. Microcarriers are a viable platform to meet the surface area requirement for generation of the predicted number of cells.

One challenge when transitioning to larger reactors is the identification of agitation conditions that allow for good suspension of the microcarriers and sufficient kLa and …

Development Of A Qualtiy Driven Integrated Continuous Biomanufacturing Process, Daniel Karst, Fabian Steinenbach, Thomas Villiger, Miroslav Soos, Massimo Morbidelli

Cell Culture Engineering XV

The recent improvements of continuous up- and downstream processes in the production of therapeutic proteins suggest their final integration to a single process stream. Besides general benefits of continuous manufacturing, such as reduced equipment size, enhanced cost efficiency and high volumetric productivity, the steady state operation favors constant or even improved product quality. However, N-linked glycosylation, charge variants, aggregates and other critical quality attributes of therapeutic proteins depend on operating conditions in up- and downstream processing. The combination of long process durations and the accompanied lack of experimental high-throughput capabilities of continuous bioprocesses demand for model based optimization regarding product …

Implementation And Evaluation Of A High-Throughput Sirna Screening System For Suspension Cho Cells, Gerald Klanert, Daniel Fernandez, Vaibhav Jadhav, Nicole Borth

Cell Culture Engineering XV

Chinese Hamster Ovary (CHO) cells are the most frequently used mammalian cell factory for the production of human-like recombinant proteins. Due to existing limitations in growth and protein production, genetic optimization of CHO cell lines may significantly enhance bioprocess productivities. Knockdown of genes by siRNAs is a standard method to identify genes involved in a desirable phenotype, either because their knockdown improves or degenerates the property. As at least 13000 different transcripts are present in a cell at any time, it is of interest to develop a method that is able to efficiently test the effect of gene knockdown at …

Efficient Production Of Influenza Virus-Like Particles In Hek-293sf Cells, Alina Venereo

Cell Culture Engineering XV

Influenza is considered a major threat to human health. The current influenza vaccines production system in embryonated eggs does not satisfy the market needs in cases of pandemic or high seasonal demand. Furthermore, even though the existent seasonal vaccines are effective to induce protective immunity in healthy adults, poor immunogenicity in the elderly and early childhood is elicited (Wu et al., 2010). Therefore, there is a need to develop new generation influenza vaccines produced in robust and flexible production platforms capable of providing complete immune protection and support the vaccines demand. Virus-like particles (VLP) constitute a promising alternative to safely …

Process Intensification Of Perfusion: To Steady-State, Or Unsteady-State, That Is The Question, Henry Lin

Cell Culture Engineering XV

Recent advances in process intensification of perfusion cell culture have diverged into two general modes of operation. One focuses on the conventional steady-state concept of maintaining constant high cell density over a lengthier culture duration, and the other focuses on a much more intensified non-steady state cell density, at times reaching >100e⁶ cells/mL. The latter has been coined concentrated fed-batch and the former continuous perfusion. Several underlying characteristics differentiate the two modes. In the steady-state approach, cell bleed is required and the cell density typically can range from 20 to 50e⁶ cells/mL. Culture duration of 30 days or …

A Multi-Omic Approach To Understanding Recombinant Protein Degradation In Chinese Hamster Ovary Cells, Ronan Kelly

Cell Culture Engineering XV

Product degradation, such as proteolytic clipping, is a common quality issue with the recombinant expression of proteins from Chinese hamster ovary cells in the biopharmaceutical industry. In this study we applied a “multi-omic” approach including RNA-seq, miRNA and proteomic analysis to identify factors contributing to the degradation of a model recombinant molecule. We identified 19 recombinant Chinese hamster ovary clonally-derived cell lines producing various levels of truncated or clipped product from a fed-batch cell culture shake flask screen. These cell lines then underwent detailed analysis using the various ‘omic approaches to identify the key causal factors linked to truncation of …

Multi-Omic Modeling Of Translational Efficiency For Synthetic Gene Design, Joseph Longworth, Javier Gonzalez, Paul Dobson, Josselin Noirel, Neil Lawrence

Cell Culture Engineering XV

Controlled expression of recombinant genes in CHO cells for advanced cell engineering will require precise, coordinated control of the synthetic processes that underpin the production of specific recombinant products or the optimal stoichiometry of functional effector proteins for multigene engineering applications. Although control of recombinant gene transcription in CHO host cells is now possible, technologies that enable control of recombinant mRNA translation rate are lacking. This is undesirable as in eukaryotic cells, cellular mRNA concentration itself may only explain a relatively small proportion of the variation in cellular protein abundance; mRNA translation rate is by far the most important contributor …

Next Gen Car T Cells, Michael Jensen

Cell Culture Engineering XV

Recent conceptual as well as technological advances in the areas of molecular immunology, gene transfer, and cell processing have fostered increasingly sophisticated translational applications of adoptive T cell therapy for oncologic disease employing genetically-modified T-lymphocytes. My laboratory’s work focuses on T-cell genetic modification for re-directing antigen specificity to tumors utilizing recent advances not only in the composition and specificity of receptor antigen recognition domains, but also the evolution of multifunctional cytoplasmic signaling domains developed for these chimeric antigen receptors (CARs) that provide dual activation and co-stimulatory signaling. My group is also investigating the context of adoptive transfer with respect to …

Scalable Production Of Mesenchymal Stem/Stromal Cells From Different Human Sources In Microcarrier-Based Stirred Culture Systems, Ana Fernandez

Cell Culture Engineering XV

Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell based therapies and the development of microcarrier-based MSC cultures in scalable stirred bioreactors combined with well-defined serum-/xenogeneic (xeno)-free medium formulations represent important milestones for the clinical-scale production of human MSC. In this work, we optimized our previously established xeno-free microcarrier-based culture system, towards the large-scale production of human bone marrow (BM)-derived MSC and adipose tissue-derived stem/stromal cells (ASC) and we have established a stirred culture system combining gelatin-based microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. In addition, we tested the ability of using …

Mab Product Consistency In Long Duration Microfiltration-Based Cho Perfusion Process, Douglas Rank, Patrick Mcinnis, Christopher Martin, Michael Phillips

Cell Culture Engineering XV

Perfusion processes have traditionally been used for the generation of unstable proteins in cell culture systems. The use of perfusion for production of stable proteins has been limited by low product concentration, media costs, and system complexity. However, with the advent of new single-use technology, cell culture media specifically formulated to support high density perfusion, and high-producing cell lines, perfusion processes are gaining widespread industry attention. Additionally, perfusion processes are considered an integral part of the “Factory of the Future” vision through enabling continuous processing while delivering a product effluent with consistent product quality and concentration.

In this study, we …

Expansion And Differentiation Of T Cells Under Defined Xeno-Free Culture Conditions, Jessie Ni

Cell Culture Engineering XV

Antigen specific suppressive immunotherapy such as Tregs therapy and highly selective targeted immunotherapy such as chimeric antigen receptor and T-cell receptor therapy have shown great benefits and therapeutic value to the patient population. These immunotherapy applications require ex vivo expansion of T cells to high cell densities without loss of T cell phenotypes and functionality for successful adoptive transfer. Moreover, a defined, xeno- and serum-free basal medium is ideal to reduce cost, limit variability and lower the risk of foreign contaminants.

By applying the quality by design approach, we report the successful development of a xeno- free basal medium containing …

A Bioinformatic Pipeline For Studying Ribosome Occupancy In Cho Cells, Shangzhong Li

Cell Culture Engineering XV

No abstract provided.

A Flow Cytometric Granularity Assay For The Quantification Of Infectious Virus, Megan Logan

Cell Culture Engineering XV

A need exists for rapid, low-cost, and accurate infectious viral quantification method in the bio-pharmaceutical field for the production of vaccines and virus-based therapeutics. Two of the most common and traditionally employed methods to quantify infectious viruses are plaque assays and cell culture infectious dose 50 (CCID₅₀); both are relatively inexpensive, however they can be time consuming and demonstrate significant variability between operators. Here we present a method to quantify infectious viruses using the post-infection granular changes within the cell using flow cytometry to create a more rapid and high throughput quantification method. To validate this method, a …

Increasing Diversity Of Production Cell Lines Through Miniaturization, Automation, And High-Throughput Analytics, Kim Le, Hannah Victor, Kristi Daris, Trent Munro

Cell Culture Engineering XV

The development of a successful biologic therapeutic manufacturing process begins with the creation of a stable clonal cell line. Since attributes of the production cell line will significantly impact upstream and downstream processes, researchers must find ways to generate several candidate lines with diverse properties. However, a wide diversity is difficult to achieve since cultures are commonly selected, maintained, and screened as populations. In these populations, robust sub-populations can overtake the overall culture and reduce diversity. To combat this, sub-populations must be physically separated by splitting or subcloning, and maintained in individual vessels requiring intensive labor and infrastructure. As a …

Use Of Quality By Design Principles For Development Of Upstream Process Control Strategy, Canghai Lu, Girish Pendse

Cell Culture Engineering XV

A systematic approach was developed using the QbD (Quality by Design) principles to study and characterize monoclonal antibody upstream production processes. This approach comprises of risk assessment of upstream process parameters, small scale model development and qualification, process characterization as well as the determination of CPPs (Critical Process Parameters) and PARs (Proven Acceptable Ranges). A high throughout analytical method to measure N-linked glycans and a Chemometric method to calibrate the results were developed in order to overcome the analytical bottleneck. These studies improved the process understanding, explored the multivariate interactions, and established the relationship between CPPs and CQAs (Critical Quality …