Engineering Commons^™

The Relevance Of Cell Size In A Cho Fed Batch Process: Metabolic And Transcriptomic Characterization, Dirk Martens, Xiao Pan, Rene Wijffels, Ciska Dalm

Cell Culture Engineering XVI

Normally the growth profile of a CHO cell fed-batch process can be divided into a growth phase followed by a stationary (non-growth) phase and a cell death phase. In this study, using a commercially available media system in a 10 liter fed-batch reactor, the growth phase is followed by a phase where cell division stops but cell growth continues in the form of an increase in cell size (Figure 1). During the cell size increase (SI) phase, the average volume and dry weight per cell increase linearly with time up to nearly threefold. Cell cycle and transcriptome analysis show that …

Curation Of A Cho Dg44 Genome Scale Model And Application To Support Cell Culture Development Process, Cyrielle Calmels, Laetitia Malphettes, Mikael Rørdam Andersen

Cell Culture Engineering XVI

Genome scale models (GSM) have become a useful tool to connect different omics dataset into a single computational framework, thus giving a good overview of the flux distribution and metabolites interconnections in a specific environmental condition. A community genome-scale metabolic network reconstruction of Cricetulus griseus and cell line specific models have been recently developed 1. The main objectives with the use of the published CHO DG44 model were to enhance industrial bioprocess performance by suggesting genetic or metabolic targets, as well as strategies for medium optimization, and by bringing more fundamental knowledge about CHO cell metabolism. In a first step, …

Bioprocess Intensification And Optimisation Using Macroscopic Predictive Models Of Cell Culture Processes, Bassem Ben Yahia, Boris Fessler, Gwendal Gränicher, An-Vy Tran, Mareike Harmsen, Elmar Heinzle

Integrated Continuous Biomanufacturing III

Chinese Ovary Hamster (CHO) are an indispensable tool for biotechnological production of biologics which is a multi-million business. Recently, the pharmaceutical industry is increasingly focusing in the use of perfusion mode. Nevertheless, the optimal perfusion rate during biopharmaceutical perfusion production is dependent on cell metabolism which can be characterized by mathematical models. This study provides insights into the predictive capacities of systematic and simple cell modeling approaches of metabolism, growth and production of monoclonal antibodies (mAb) [1] to optimized medium composition and perfusion rate during CHO perfusion culture. We applied the metabolic steady state concept and used a segmented linear …

Use Of A Biphasic Perfusion Process Based On Mild Hypothermia For Recombinant Glucocerebrosidase (Gba) Production, Filipa Gonçalves, Juliana Coronel, Leda R. Castilho

Integrated Continuous Biomanufacturing III

The main goal of this study was to develop an innovative CHO-based process for the production of glucocerebrosidase (GBA), an enzyme used for the replacement therapy of Type 1 Gaucher disease. The focus of the present study was on the development of a perfusion process, combining strategies that are commonly used for process optimization: temperature reduction, and supplementation of the culture medium with productivity enhancers, such as short chain fatty acids. The effects of mild hypothermic conditions combined with valeric acid supplementation were first studied in batch shake flasks for two clones (CHO-GBA-36K and CHO-GBA-65P), developed previously using as host …

Clarification And Capture Of A Cho-Derived Monoclonal Antibody Through Flocculation And Aex Processes, Rimenys J. Carvalho, Leda Castilho

Integrated Continuous Biomanufacturing III

Flocculants have been widely used in the food and chemical industry, as well as in wastewater clarification. More recently, studies have shown the applicability of these agents in the clarification of mammalian cell suspensions as an alternative e.g. to centrifugation and microfiltration1,2. Flocculants present several advantages such as low cost, process simplicity and facility to run under continuous mode. On the other hand, some flocculants have shown toxicity and thus their total clearance has to be demonstrated. In this work, the cationic flocculants chitosan and poly diallyldimethylammonium chloride (PDADMAC) have been investigated and compared for the clarification of suspensions of …

Bioprocess Intensification And Optimisation Using Macroscopic Predictive Models Of Cell Culture Processes, Bassem Ben Yahia, Mareike Harmsen, An-Vy Tran, Gwendal Gränicher, Boris Fessler, Elmar Heinzle

Integrated Continuous Biomanufacturing III

CHO cells, modeling, perfusion, ATF, segmented linear model

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 …

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 …

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, …

Integration Of Transcriptomic Data With A Genome-Scale Model Reveals Key Metabolic Features Of High Producer Cho Cell Lines, Natalia Jimenez

Cell Culture Engineering XV

No abstract provided.

Small-Scale Comparison Of Pseudoperfusion Feeding Strategies Using Basal And Concentrated Feed Media, Iona Bettinardi, Leda Castiho

Cell Culture Engineering XV

Perfusion has long been the industrial choice for the production of unstable proteins, and nowadays is being intensively studied also for stable proteins due to its ability to keep high viable cell densities and high volumetric productivities over long operation times. However, conventional perfusion is fed with basal medium at high dilution (or perfusion) rates, causing a dilution of the protein of interest (POI) and generating large volumes of harvest to be processed in the purification steps.

In this work, small-scale pseudoperfusion experiments were performed to compare the conventional perfusion strategy using basal culture medium (TC-LECC, Xell AG) with a …

Systems Analysis Of Cho Cell Metabolism For Enhanced Fed-Batch Process Performance: Identification Of Novel Growth Inhibitors And Their Control, Bhanu Chandra, Jaitahree Kale, Anamaria Ovalle, Gregory Hiller

Cell Culture Engineering XV

Over the past decade, process development efforts have led to attaining titers close to 3-4g/L on a routine basis in CHO fed-batch cultures. However, with the advent of biosimilars and the ever expanding therapeutic portfolio of innovator drugs, there is a growing need for enhancing the throughput of fed-batch cultures in order to meet the product requirements with the limited manufacturing capacity. The classical hurdle for higher productivities in fed-batch cultures is the sub-optimal metabolism of mammalian cells that causes accumulation of byproducts, including lactate and ammonia, resulting in growth inhibition. With a robust CHO expression system, our group has …

Challenges Of Scale Down Model For Disposable Bioreactors: Case Studies On Growth & Product Quality Impacts, Jincai Li, Guangming Zhang, Huilin Zhu, Weichang Zhou

Cell Culture Engineering XV

Despite wide-spread use of disposable bioreactors, there is a lack of well-established scale-down model for larger scale SUBs. Here we report a case of NS0 cell culture process transfer from 2000L stainless steel bioreactor (SST) to 2000L disposable bioreactor (SUB). Initial attempts in trying to grow the NS0 cells in the small scale 2D bags yielded non-satisfactory results, as growth was impacted by bag material type as well as by suppliers of the same bag material type. However, 3D bags of 50L and above proved to be supportive of the NS0 cell line growth.

Even for cell lines that do …

Assessment Of Genomic Instability In Chinese Hamster Ovary (Cho) Cells, Sabine Vcelar, Martina Baumann, Michael Melcher, Nicole Borth

Cell Culture Engineering XV

CHO cells are the number one production system for therapeutic proteins due to their ease of handling, their fast growth in suspension culture and their capability to perform complex protein folding and human-like post-translational modifications. This flexibility is in part due to, but at the same time set off by the frequent occurrence of chromosomal rearrangements and other genomic variants, which influences individual cell line performance and the stability of industrial producer cell lines, resulting in prolonged screening phases in order to isolate cells with sufficiently stable properties. Furthermore producer cell properties are also frequently lost again over time and …

Reduction Of Metabolic Waste Products, Ammonia And Lactate, Through The Coupling Of Gs Selection And Ldh-A Down-Regulation In Cho Cells, Soo Min Noh, Jin Hyoung Park, Gyun Min Lee

Cell Culture Engineering XV

The cultivation of Chinese hamster ovary (CHO) cells for the production of therapeutic proteins inevitably accompanies the production of metabolic wastes, mostly ammonia and lactate. Ammonia alters cell growth, productivity and the glycosylation patterns of proteins, and lactate acidifies culture media, having negative effects on cell culture. A stable CHO cell line should be established for the manufacturing process of therapeutic proteins, and the development of stable cell lines is usually based on two expression systems: the dihydrofolate reductase (DHFR) system and the glutamine synthetase (GS) system. Compared to the DHFR system, the GS system produces a reduced level of …

Challenges Of Scale Down Model For Disposable Bioreactors: Case Studies On Growth & Product Quality Impacts, Jincai Li, Guangming Zhang, Huilin Zhu

Single-Use Technologies: Bridging Polymer Science to Biotechnology Applications

Despite wide-spread use of disposable bioreactors, there is a lack of well-established scale-down model for larger scale SUBs. Here we report a case of NS0 cell culture process transfer from 2000L stainless steel bioreactor (SST) to 2000L disposable bioreactor (SUB). Initial attempts in trying to grow the NS0 cells in the small scale 2D bags yielded non-satisfactory results, as growth was impacted by bag material type as well as by suppliers of the same bag material type. However, 3D bags of 50L and above proved to be supportive of the NS0 cell line growth.

Even for cell lines that do …

Case Study: Identification, Characterization And Mitigation Of Growth Inhibitory Leachable Compound From Disposable Plastic Vessels, Zara Melkounian

Single-Use Technologies: Bridging Polymer Science to Biotechnology Applications

The inhibition of CHO cell growth was observed in culture medium incubated in disposable plastic vessel, but not in a control glass vessel. Gamma irradiation of vessels during terminal sterilization step was required to produce the growth inhibitory leachable, as unsterilized vessels showed no inhibition of cell growth. Also, the growth inhibition was cell line specific in that some CHO cells were more sensitive than others, and some were not sensitive at all.

Using chromatographic and spectroscopic methods we were able to identify and characterize a single leachable responsible for this growth inhibition in some CHO cells. By understanding the …

Impact Of A Toxic Single-Use Bio-Process Container Film Leachable On Chinese Hamster Ovary Cells, Heather Nunn, Matthew Hammond

Single-Use Technologies: Bridging Polymer Science to Biotechnology Applications

A high-throughput cell-based assay (HT-CBA) was developed to investigate the impacts of the polymer films used to construct single-use bio-processing equipment such as bio-process containers (BPCs) and single-use bioreactors (SUBs) on the growth and health of Chinese hamster ovary (CHO) cells. Cell growth media were incubated in BPCs constructed from several different films, and the media thus treated was used to culture CHO cells in 24 deep-well plates. Multiple films negatively impacted culture growth and viability. The compound bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) which leaches at low levels from these films was shown to be highly detrimental to cell growth. The toxic compound …