Engineering Commons^™

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 …

Targeting Product Quality: Where Systems Biotechnology And Process Design Meet, Markus Michael Muller, Jan Bechmann, Ingo Gorr, Harald Bradl, Jan Visser

Cell Culture Engineering XV

Product quality is a result of the entire production process including protein sequence, host cell, media and process parameters. Many of the desired product properties are defined by posttranslational modifications with impact on biological activity, immunogenicity, half-life or stability. In‑depth understanding of the host cells capabilities as well as of the process interactions enables the targeted modulation of product quality attributes by rational selection of host cells and design of bioprocesses. This is valuable for new biological molecules in order to improve efficacy, reduce side effects, access new patient populations. For biosimilars this allows developing into defined quality attribute profiles. …

Perfusion Media Development And Evaluation With Spin Tube And Ambr15 Highthroughput Small-Scale Models, Yang Wang, Shawn Barrett, Borka Naumovich, Jessica Nichols, Steve Gorfien

Cell Culture Engineering XV

For over 75 years, continuous manufacturing has been investigated in both academia and industry as an alternative to batch manufacturing. Perfusion is a well-recognized method for continuous production of biomolecules expressed in CHO cell culture and it is known to have certain advantages over batch and fed‑batch processes. The major advantage of perfusion processes is that a constant environment is provided to cells through continuous by-product removal and nutrient addition. However, it has proven to be challenging to apply high-throughput approaches routinely used in fed-batch process development while maintaining true continuous culture, and so semi-continuous methods have instead been pursued …

A Community Genome-Scale Model Of Chinese Hamster Ovary Cell Metabolism Identifies Differences In The Efficiency Of Resource Utilization For Various Bioprocesses, Hooman Hefzi

Cell Culture Engineering XV

Genome-scale models of metabolism have successfully been employed in many microbial and eukaryotic metabolic engineering efforts by guiding pathway engineering and media optimization. They have also been used to explore the genotype-phenotype relationship in mammalian cells. The publication of the genomic sequence for Chinese hamster ovary (CHO) cells has allowed generation of genome-scale metabolic models (GeMs) for this organism. Here we have developed a high-quality community CHO GeM via careful reconciliation and manual curation of three independently developed CHO GeMs. This metabolic model, consisting of over 4000 metabolites and 6000 reactions, is capable of integrating proteomic, transcriptomic, and metabolomic data …

Fluxomics: The Integration Of Metabolic Flux Analysis (Mfa) With Multivariate Data Analysis (Mvda) To Identify Key Process Parameters For Cho Cell Culture, Neil Templeton

Cell Culture Engineering XV

An integrated ¹³C-metabolic flux analysis (MFA)/fluxomics approach was conducted to characterize fed-batch, concentrated fed-batch, and perfusion modes of operation. While both the concentrated fed-batch and perfusion processes were independently represented by one metabolic quasi-steady-state, fed-batch metabolism was characterized by multiple. Intracellular flux maps were developed for all three operational modes. To elucidate the phenotype of peak specific productivity, the stationary phase of fed-batch was characterized by ¹³C-MFA. The metabolic network included glycolysis, the pentose-phosphate pathway, citric acid cycle, and various anaplerotic reactions. Anabolic demands for biomass, host cell protein (HCP), and IgG were accounted for. To identify potential …

Cell Therapy Manufacturing Strategies: Impact On Cost Of Goods, Cost Of Development And Commercialisation, Suzanne Farid

Cell Culture Engineering XV

Successful commercialisation of cell therapies will be underpinned by cost-effective, robust and scalable manufacturing processes, practical supply chain solutions for delivery to patients and early planning of reimbursement. UCL’s Decisional Tools team have developed advanced decision-support tools that effectively integrate concepts from bioprocess economics, risk analysis, and combinatorial optimization to address such challenges. This presentation will provide some of our most recent process economic insights from such models applied to current and future cell therapy manufacturing processes. Bottlenecks in upstream and downstream processes will be identified and the technical innovation required to bridge the gaps constraining commercialisation will be discussed. …

Human Cardiac Stem Cells For Allogeneic Cell Therapies: Integrating Bioprocess Development And ‘Omics Characterization Tools, Patricia Gomes

Cell Culture Engineering XV

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Myocardial infarction (AMI) severely affects patients’ heart muscle and microvasculature, critically decreasing the number of functional cardiomyocytes (CM). Stem cell and protein based therapies became promising cardiac repair strategies since it was found that under pathological stress, resident cardiac stem cells (CSC) of the adult myocardium are activated by growth factors (GF) secreted by the surviving CM. Consequently, an auto/paracrine loop is triggered to maintain GF production, which enhances CSC activation and differentiation into new CM, endothelial and smooth muscle cells contributing to the repair of damaged myocardium [1]. Since …

Pcl-Pdms-Pcl Copolymer-Based Microspheres Mediate Cardiovascular Differentiation From Embryonic Stem Cells, Yan Li

Cell Culture Engineering XV

Poly-ϵ-caprolactone (PCL) based copolymers have received much attention as drug or growth factor delivery carriers and tissue engineering scaffolds due to their biocompatibility, biodegradability, and tunable biophysical properties. Copolymers of PCL and polydimethylsiloxane (PDMS) also have shape memory behaviors and can be made into thermoresponsive shape memory polymers for various biomedical applications such as smart sutures and vascular stents. However, the influence of biophysical properties of PCL-PDMS-PCL copolymers on stem cell lineage commitment has not been well understood. In this study, PDMS was used as soft segments of varying length to tailor the biophysical properties of PCL (hard segments)-based co-polymers. …

Utilizing Rna-Seq Technique To Improve Molecular Understanding Of Chinese Hamster Ovary (Cho) Cell Bioprocessing, Yogender Kumar, Christpher Saski, Sarah Harcum

Cell Culture Engineering XV

Chinese Hamster Ovary (CHO) cells are an important biopharmaceutical cell line, accounting for the production of over 70% of the approved protein therapeutics. However several limitations exist with the use of CHO cell lines including low product titers. Understanding of CHO cells in bioprocessing has up until now relied heavily on empirical results with a limited knowledge of the intracellular dynamics. With the recent establishment of both Chinese hamster and CHO-K1 cell line genome assemblies, it is now possible to leverage the genomic resources to better understand and further improve CHO cell bioprocessing. In this study, RNA-Seq, next-generation transcriptome sequencing, …

Scale-Down And Initial Characterization Studies Of An Allogeneic Cell Therapy Manufacturing Process, Thomas Brieva

Cell Culture Engineering XV

Cancer therapies leveraging the immune system have been improving the course of disease. T-cell therapies are one of the modalities of immune therapy, which also include small molecules, proteins, and various types of immune cells. T-cell therapies include T-cell Receptor cells (TCR), Chimeric Antigen Receptor (CAR) T-cells, and Cytotoxic T-Lymphocytes (CTL). Although the nascent cell therapy industry faces many manufacturing challenges, it benefits from the vast experience of the blood industry and cell-produced biologics industry.

A notable manufacturing challenge for most T-cell therapies results from their autologous nature, in which cells from a patient are used to produce the therapeutic …

Multi-Omic Profiling Of Epo Producing Cho Cell Panel Reveals Metabolic Adaptation To Heterologous Protein Production, Daniel Ley

Cell Culture Engineering XV

The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed a multi-­omic physiological characterization including metabolic footprinting of amino acids, …

Michael Borys (Bristol- Myers Squibb) Incorporation Of Qbd Elements Into The Development And Characterization Of A Second Generation Process, Michael Borys, Nicholas Abu-Absi

Cell Culture Engineering XV

QbD principles are readily incorporated into mammalian cell processes to streamline process development and characterization. A key enabler of the implementation of these principles has been widespread adoption of platform technologies by the industry. This allows easy and efficient navigation of the QbD roadmap laid out in the A-Mab case study over the course of the development lifecycle of a product.

Here we examine the case of a 2nd generation process for a legacy product that was originally developed and approved using the traditional approach to process development and characterization. The goal of the 2nd generation process was to achieve …

Manipulation And Exploitation Of Micrornas For Enhanced Recombinant Protein Production In Cho Cells, Tulshi Patel, Lyne Josse, Mark Smales, Robert Young

Cell Culture Engineering XV

MicroRNAs (miRNAs) are regulatory non-coding RNA molecules around 22 nucleotides in length which control gene expression via translational repression of their cognate messenger RNA(s) (mRNAs). Regulating as much as 30% of the genome, miRNAs exhibit a high level of promiscuity as a single miRNA is able to target multiple mRNA targets and a single mRNA transcript can be under the regulation of several miRNAs. Thus, global regulation and control of almost all cellular processes and pathways is heavily dependent on the level and activity of miRNAs. This work has set out to investigate the biology of miRNAs in the industrially …

Development Of Plate-Based Sialic Acid Assays To Support Clone Screening And Early Stage Upstream Process Development, Julie Gardin, Yvette Tang

Cell Culture Engineering XV

Sialic acid, a post-translational modification, is an important product quality attribute of glycoproteins. The levels of sialic acid may impact solubility of the final product and the half-life (product clearance). In addition, the N-glycolylneuraminic acid (NGNA) form of sialic acid may elicit an immunogenic response in humans. In an effort to have a more high-throughput evaluation of these product quality attributes, two plate based assays were developed.

Specifically, a direct NGNA ELISA was developed using a polyclonal chicken anti-NGNA antibody. This assay was implemented as one of the clone screening tools to eliminate clones that may produce more of the …

Biotherapeutic Development In The 'Omics Age: The Cho Genome And Beyond, Chapman Wright, Joost Groot, Scott Estes

Cell Culture Engineering XV

The Next-Generation Sequencing (NGS) revolution has had a profound effect on the manner in which we approach the fields of biology and medicine, but the effects are not limited to these fields alone. With the process of collecting and analyzing large datasets becoming more seamless, a growing number of disciplines are incorporating NGS and other ‘Omics technologies into their workflow. Biotherapeutic development is one such field that is benefitting from the comprehensive and rich datasets that NGS can provide. At Biogen, we are using NGS in two ways: to provide a deeper understanding CHO cell biology during bioprocessing and to …

Fluorescent Influenza-Like Particles And Control Over Their Composition, Marc Aucoin

Cell Culture Engineering XV

The Baculovirus Expression Vector System (BEVS) is a proven platform for making biologics. Despite being widely used, the system has typically relied on ‘brute force’ high expression. Building off of the handful of scientific publications that have shown that alternate promoter usage can promote higher functional protein yields, we have sought to generate baculovirus constructs that make use of a greater variety of promoters and genetic elements (shRNA/CRISPRi) to better control gene expression.

Materials and Methods

Genes for green fluorescent protein (GFP), DsRed2, and iRFP682, alone or as fusions to the human influenza A/PR/8/34 hemagglutinin (HA), matrix (M1) and neuraminidase …