Biomedical Engineering and Bioengineering Commons^™

Building Qbd Frameworks Retrospectively For Commercial Products And The Use Of Scale-Down Model Qualification Strategies To Support Continuous Improvement, Jose Menezes, Francisca Gouveia, Pedro Felizardo

Cell Culture Engineering XV

A science-based justification for many continuous improvement and post-approval submissions on ‘legacy products’ (i.e., commercial products developed without a QbD rationale) requires that some of those missing QbD elements in the original filing, be defined retrospectively.

Here we present a new proposal to achieve that, that is general and very capable of supporting the transition of ‘legacy’ non-QbD into quasi-QbD filings. It is based on (1) a formal criticality analysis to the current process version to support the choice of quality attributes and process parameters in the filing, and identification of additional ones that may impact the quality target-product profile …

Separation Of Igg Glycoforms For Biosimilars Development Using Fc Gamma Receptors As Affinity-Based Chromatography Ligands, Austin Boesch

Cell Culture Engineering XV

Abstract: In biosimilars development, matching the glycosylation pattern of the innovator molecule can be a significant challenge in cell culture development. Here we present a novel downstream method using Fc gamma receptors as chromatography affinity ligands to separate monoclonal antibody glycoforms. Additional data is presented highlighting glycoform potentiation of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis which can be important mechanisms of action to consider in biosimilars development

Improvement Of Cho Specific Productivity Using Amino Acid Derivatives, Aline Zimmer, Caroline Hecklau, Sascha Pering, Alisa Schnellbaecher, Thomas Eichhorn

Cell Culture Engineering XV

Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Amino acids are key elements for the cellular metabolism but also for the quality of the recombinant protein produced. Several studies have already demonstrated the link between amino acid availability and sequence variants [1-3] or specific modifications like trisulfide bonds [4]. To avoid such modifications, the chemical modification of amino acids is an interesting alternative to modulate their overall solubility [5], …

Development Of A Chemically Defined Media And A Chemically Defined Feeding Strategy For Extended Growth And Enhanced Productivity In Cho-K1 And Cho Dg44 Cultures, Sagar Kokal, Kyle Liu, John Menton

Cell Culture Engineering XV

The biopharmaceutical industry utilizes Chinese Hamster Ovary (CHO) cells for the production of recombinant proteins. Due to regulatory restrictions, there has been much focus on using serum-free culture processes including chemically defined (CD) media and feed system. A great deal of time and cost has been invested by biopharmaceutical companies for the development of an optimal CD media. Currently, the fed-batch process is the most dominant method for the large cultivation of CHO cells. In response to these industry requirements, Kerry has developed a complete system including a CD media called Sheff-CHO CD complete and a CD feed system named …

The Differential Polarizability Of Cho Cells Can Be Used To Monitor Changes In Metabolism, 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 …

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 …

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 …

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 …

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 …