Engineering Commons^™

Scalable Lentiviral Vector Production Using Stable Producer Cell Lines In Perfusion Mode, Sven Ansorge, Aziza Manceur, Stéphane Lanthier, Sonia Tremblay, Anne-Marie Gélinas, July Dorion-Thibaudeau, Sophie Broussau, Julia Transfiguracion, Hafida Aomari, Rénald Gilbert

Cell Culture Engineering XVI

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies against genetic and acquired diseases, as well as in cancer therapies. To address the challenges linked to the generation of preclinical and clinical supply, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. We focus on the development of perfusion processes to both intensify the process and harvest produced LV …

Dxb11/Taut Host Cell Engineering Strategy Enabling The Establishment Of Strains Producing The Highest Yield Of Advanced Recycling Antibodies, Hisahiro Tabuchi, Masaki Yamaguchi, Tao Jialin, Hirokatsu Makitsubo

Cell Culture Engineering XVI

Innovation in monoclonal antibody (mAb) production for clinical use continues to be driven by cell engineering strategies to increase mAb yield and to control the complexity of advanced recycling antibodies (rcAbs). rcAbs offer significant advantages for efficacy by dissociating the antigens in endosomes and recycling free antibodies back to plasma. We were able to reduce the instability in an rcAb-producing CHO cell line whereby mAb titer during the stage of cell line development (CLD) was decreased and improve cell culture optimization. In this study, we used a chemically defined medium (CDM) and CDM-adapted transporter-overexpressing DXB11 host cells for CLD of …

Novel Transposase Tools For Cell-Line Engineering, Ferenc Boldog, Jeremy Minshull, Mark Welch, Kate Caves

Cell Culture Engineering XVI

ATUM has discovered, characterized, engineered and patented new transposases that work comparable to, or better than previously commercialized transposases. The technology is highly valuable for protein expression and genome engineering applications. It enables a specified sequence to behave as a transposon, a mobile genetic element, which can efficiently transpose between vectors and chromosomes via a “cut & paste” mechanism. The Leap-In Transposase® catalyzes the integration of a transposon containing your gene into TTAT sites in the target genome. During transposition, the Leap-In Transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and moves …

Empowering Manufacturing Decisions Through Process Simulation Models, Tiffany Rau, Terence Davidovits, Rick Stock

Cell Culture Engineering XVI

The need for Clinical and Commercial manufacturing capacity continues to increase and at the same time there are “shortages” of production slots and time pressures. Whether one is a virtual, small or large company there are many considerations that go into the decision of how and where to manufacture the biopharmaceutical, cell therapy or vaccine. In this presentation, we explore approaches for using commercially available modeling tools, such as Super ProDesigner® and BioSolve™, to simulate the manufacturing of a “typical” monoclonal antibody product and a multivalent conjugate vaccine product. Through these hypothetical case studies, we will illustrate several important concepts …

Conference Program, Anne Skaja Robinson, Raghavan Venkat, Gene Schaefer

Cell Culture Engineering XVI

No abstract provided.

Impact Of Raw Materials On Sialyation For A Therapeutic Protein, Wei-Chien Hung, Mei Shao, Bridget Leslie, John Mavrianos, Paul Loray, Mei Shao

Cell Culture Engineering XVI

Total sialic acid content (TSAC) is a critical product quality attribute (CQA) for a therapeutic protein. Previous studies and results have suggested lot-to-lot variability of raw material, media powder and feed, have potential effects on total sialyation acid content (TSAC) at cell culture fluid (CCF) and harvested cell culture fluid (HCCF). Through searching literature, we found that potential components in media powder and feed such trace metal and vitamins can greatly modulate the sialyation acid level in vivo or in vitro. For instance, the sialic acid content was simultaneously increased in glycopeptides as well as gangliosides under lithium treatment^{1 …}

Demonstrating A Powerful Scale-Up Strategy For Biosimilar Mab In Single Use Systems Via Physicochemical And Functional Characterization, Serdar Alpan, Ozge Can, Deniz Baycin, Yigit Erdemgil, Zeynep Yildirim Keles, A. Emin Atik

Cell Culture Engineering XVI

Biosimilars have received a remarkable attention in the recent years. Due to the heterogeneity of biosimilar mAbs, they need to be well-characterized by various orthogonal techniques in order to identify their physicochemical and functional characteristics. Characterization of the post translational modifications, especially, glycosylation is vital to define the critical quality attributes (CQAs) which affect safety, efficacy and quality of drugs. In this study, we were able to manipulate the quality of the drug by using scale-up strategies for single use systems. By using ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS), we were able to demonstrate physicochemical similarities between …

The Journey From Tech Transfer To Bla Submission: Case Study Of A Ns0 Cell Culture Process From 2000l Stainless Steel Bioreactor To 2000l Disposable Bioreactor, Jincai Li

Cell Culture Engineering XVI

A case study of NS0 cell culture process transfer from 2000L stainless steel bioreactor (SST) to 2000L disposable bioreactor (SUB), and through to process validation and BLA submission is reported for production of an antibody therapeutics in this poster. Initial attempts in growing 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 different suppliers of the same bag material type. However, 3D bags of 50L and above proved to be supportive of the NS0 cell line growth. Process characterization (PC) and process validation (PV) efforts …

Identifying Hipk1 As A Target Of Mir-22-3p Enhancing Recombinant Protein Production From Hek 293 By Using Microarray And Htp Sirna Screen, Sarah Inwood, Eugen Buehler, Michael Betenbaugh, Madhu Lal, Joseph Shiloach

Cell Culture Engineering XVI

Enhancing protein production in mammalian cells is of interest in the biomedical field for a variety of reasons, including structural studies and antibody production. Using small non-protein coding RNA such as microRNA has recently been a promising method of increasing protein expression. A high throughput human microRNA screen in HEK 293 cells previously identified miRNA 22-3p as a promising candidate for increasing recombinant protein expression. This microRNA enhanced the expression of luciferase, two hard-to-express membrane proteins and a secreted hFc-fusion protein. In order to explore the mechanisms of this increase in protein production and to understand the intracellular events, we …

Towards Model-Based Bioprocess Characterization: A Mathematical Model Of Cell Cycle, Metabolism And Apoptosis Of Mab-Producing Mammalian Cells, António Lima Grilo, Athanasios Mantalaris

Cell Culture Engineering XVI

The biologics industry is changing and all players seek competitiveness through process optimization aiming at satisfying the market demand providing a compliant protein in as short time as possible. As blockbuster drugs experience patent expiry, several biosimilars are coming market and competition becomes fiercer. Unlike classical experiments-based optimization, bioprocess modeling is a rational, economically efficient and reliable alternative to deliver an optimized bioprocess. The biological aspects of a bioreactor are diverse and include cells growing and cycling, other cells dying, nutrients being consumed and by-products accumulating, energy production and usage and therapeutic proteins being produced. The understanding of the biology …

Impact Of S-Sulfocysteine On Fragments And Trisulfide Bond Linkages In Monoclonal Antibodies, Aline Zimmer, Ronja Seibel, Alisa Schnellbaecher, Susanne Bohl, Maria Wehsling, Sandra Maier, Anne Zeck

Cell Culture Engineering XVI

The quality of recombinant proteins such as monoclonal antibodies produced using Chinese hamster ovary cell-based mammalian systems is dependent on many factors, including cell line, process and cell culture media. Due to these factors, the generated product is heterogeneous and may have chemically induced modifications or post-translational modifications that affect antibody stability, functionality and, in some cases, patient safety. This study demonstrates that S-sulfocysteine, a cysteine derivative, can increase the antibody specific productivity in different cell lines cultivated with different processes while minimizing trisulfide linkages in generated mAbs, mainly between heavy and light chain. The supplementation of a cell culture …

Strategies To Improve Productivity Of Cho-S Cells Expressing An Anti-Tnfα Monoclonal Antibody With Biosimilar Potential, Ana M. Moro, Matheus Luchese, Lilian Tsuruta, Wagner Quintilio, Mariana Lopes Dos Santos

Cell Culture Engineering XVI

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. This project is based on the first development stage for an adalimumab biosimilar candidate with potential for national production through the generation of a productive and stable cell line and …

Off-Target At-Scale Scale Down Model Verification Of A Marketed Biopharmaceutical, Markus Emmler, Stefanie Menrad, Yosuke Watanabe

Cell Culture Engineering XVI

According to authority guidelines, process understanding is considered the key factor for a successful control strategy in submissions for biopharmaceutical production processes. This knowledge is usually gained in scale down model (SDM) experiments and therefore is subject to the ability of the SDM to reliably predict behavior of cell culture processes in manufacturing scale. In order to prove predictability, SDM runs at target conditions are typically compared to manufacturing scale target runs only. At the same time, predictability is also assumed within the whole design space described or even beyond as evaluated in process design studies. The proof at off-target …

Development Of A Platform Expression System Using Targeted Integration In Chinese Hamster Ovary Cells, Scott Bahr, Trissa Borgschulte, Michael Johns, Jeff Galligan

Cell Culture Engineering XVI

In recent years the biomanufacturing industry has seen significant improvements in recombinant protein production titers due to advancements in protein expression technologies as well as media, feed and manufacturing process development. However, the standard methods of recombinant cell line development have remained relatively unchanged. The majority of the biopharma industry introduces transgenes into Chinese Hamster Ovary (CHO) cells using mechanical or chemical transfection processes followed by metabolic or antibiotic selection of stable recombinant pools. Through this process, the transgene(s) are randomly integrated into the genome, often times resulting in significant heterogeneity within the stable pools. Individual recombinant CHO cells within …

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, …

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 …

Tools And Methods For Providing Assurance Of Clonality For Legacy Cell Lines, Paul Wu, Juhi Ohja, John Thrift, Taymar Hartman, Louise Almond, Jennitte Stevens, Sam Yaghmour, Christina Alves, David Shaw, Mike Laird, Robyn Emmins, Yuan Zhu, Ren Liu, Zhimei Du, Rolf Koehler, Thomas Jostock, Karin Anderson, Howard Clarke, Chris Campbell

Cell Culture Engineering XVI

Over the last several years demonstration of cell line clonality has been a topic of many industry and regulatory presentations and papers. Guidance has been provided by the regulatory authorities, especially the FDA, on a path forward for providing evidence of clonality with high probability. It has been recommended that two-rounds of limiting dilution cloning (LDC) at sufficiently low seeding densities (≤0.5 cells/well) provides sufficient evidence that a cell line is clonal. Furthermore, one-round of LDC may also suffice if supplemental data from a characterized FACS or plate-imaging workflow are also included in the package.

Cell lines generated by methods …

Geoscan-M Use At Ma’Aden Phosphate Al Jalamid Mine, Saudi Arabia, Luke Balzan, Zoran Bauk, Andrew Harris

Beneficiation of Phosphates VIII

Access to real-time data is critical for process control. Scantech’s GEOSCAN-M elemental analyser has been utilized in the minerals industry for the past two decades in a range of different commodities. Already established for use in the coal and cement industries, commodities such as iron ore, copper, lead zinc, manganese, bauxite and lithium have benefited from the real-time access to elemental composition data on conveyed flows provided by the GEOSCAN. The phosphate industry has also recognized the benefits of the equipment and a number of operators and companies have installed GEOSCANs to provide real-time analysis back to the plant.

A …

Can Your P2o5 Be Commercially Exploited?, Ewan Wingate, Michael E. Kelahan, M. Robert Kelahan

Beneficiation of Phosphates VIII

World reserves of high-grade, high-quality phosphate rock are steadily decreasing. As a result, lower quality rock is being increasingly evaluated to determine its potential for commercial exploitation. It is well known that the chemical and mineralogical composition of phosphate rock varies widely from deposit to deposit, and therefore, the conditions for processing different phosphate rock types also varies.

Unfortunately, most early stage phosphate feasibility studies tend to focus on the P₂O₅ content and neglect the other chemical constituents or impurities in the rock. It is the remainder of the chemical constituents that often define the quality of …

Milling Of The Phosphate Rock Flotation Circuit Circulating Load Aiming Production Increase And Iron Content Reduction In The Final Concentrate, André Carlos Silva, Michelle Fernanda De Lira Teixeira, Anastácio Honório De Melo Filho, Thiago Drumond De Alvarez De Araújo, Maurício José De Oliveira Júnior, Wanderson Ferreira Borges Júnior, Bruno Palhares Milanezi, Jailson Pinto Cardoso

Beneficiation of Phosphates VIII

Phosphorus has its main application in the production of phosphoric acid, being the apatite series its main ore. The Brazilian main phosphorus reserves are igneous, as is the case of Catalão alkaline dome, were Copebras/CMOC International has operations. The content of P₂O₅ in the ROM has been reduced over the years and, as a consequence, the content of the contaminants in the concentrate, produced by flotation, which enhances the difficulty in the phosphoric acid production. Only traces of arsenic, iron, and heavy metals can be present in high-purity phosphoric acid. The circulating load of the Copebras’ phosphate …

Revisiting The Merseburg Process: Economic Opportunity And Environmental Benefit?, Gary Albarelli

Beneficiation of Phosphates VIII

The process for manufacture of ammonium sulfate from gypsum, known as the Merseburg Process, named for the town in Germany where it was developed, has been commercially produced in plants in Austria, India, Pakistan and the United Kingdom in the past. It is based on reacting ammonia and carbon dioxide to produce ammonium carbonate, which is then reacted with gypsum to produce ammonium sulfate and calcium carbonate. Ammonium sulfate is mainly used as direct fertilizer or in producing compound fertilizers. The global ammonium sulfate market is projected to grow 4% annually through 2025, from $4.6 billion to $6.8 billion. As …

Influence Of The Impeller Speed On Phosphate Rock Flotation, André Carlos Silva, Elenice Maria Schons Silva, Fernanda Santos Andrade

Beneficiation of Phosphates VIII

Flotation represents, at present, the technology capable of efficiently concentrate the minerals of interest, separating them from other gangue minerals. The flotation process is based on interactions between minerals (solid phase), water (liquid phase), and air (gas phase). However, it depends on physical forces driving the system. If mineral particles do not have enough momentum during the collision with the air bubble no attachment between them occurs, and consequently no flotation. Impeller rotation speed is undoubtedly a factor that changes the hydrodynamic conditions of the flotation system playing a vital role on it. It supplies the flotation system with air …

Latest Trend For Fluorine In The Phosphoric Industry: Absorption Efficiency Improvement, Conversion Into Raw Material, Tibaut Theys

Beneficiation of Phosphates VIII

Almost all the phosphate apatite used in the world to produce phosphoric acid trough the wet process contains fluorine. When not properly controlled, this element creates process, health and environmental issues that can have dramatic impact. In recent year several technologies have been established or improved to tackle these challenges.

F absorption has been improved by simple changes that ensure a high efficiency while minimizing the CAPEX.

F usage has also been largely investigated to produce valuable product as AlF3 or HF but also easy to transport raw material as CaF2. This paper will describe some recent development related to …

Alternative Depressor For Apatite Flotation, André Carlos Silva, Débora Nascimento Sousa, Diego Valentim Crescente Cara, Alex Malue Machado, Elenice Maria Schons Silva

Beneficiation of Phosphates VIII

The reality of the phosphate mines in the world has been changing. The reserves content has reduced over the years. As a consequence of this reduction, contaminants such as iron, silica and magnesium contained in phosphate rock concentrate are affecting the stability of the chemical plants, which compromises the rate of phosphate utilization and the quality of the products produced. In order to obtain a concentrate of phosphatic rocks of market acceptable quality (35% P₂O₅), the flotation process is the most suitable method for processing this. The present study aims to evaluate the potential of replacing …

The Surface Chemistry And Microflotation Of Xenotime And Depression Of Gangue Minerals, Corby Anderson, Yicheng Zhang

Beneficiation of Phosphates VIII

A pre-concentrated xenotime sample and four selected gangue minerals, ilmenite, zircon, schorl and staurolite were used in this project. Using Octano-hydroxamic acid as a collector, the surface chemistry was investigated through surface area measurements, zeta potential tests and adsorption density determinations. The results of adsorption studies were then correlated with microflotation tests conducted at room temperature in a Partridge-Smith cell. In this paper, the surface chemistry and microflotation behaviors are then discussed based on both lab observations and a literature review.

Further, because of their selective depressing power, sodium silicate and lignosulfonate have been widely used as depressants in rare-earth …

Using Two Alternative Vegetable Oil’S As Collector In Apatite Froth Flotation, André Carlos Silva, Elenice Maria Schons Silva, Alex Malue Machado, Diego Valentim Crescente Cara, Débora Nascimento Sousa

Beneficiation of Phosphates VIII

The Brazilian phosphate deposits are of igneous origin exhibiting a complex mineralogy where fluorapatite is the predominant mineralogical variety fron the apatite group, this deposits still contain carbonateapatite and hydroxyapatite associated with secondary phosphates, iron oxides (magnetite, goethite and limonite), micas and minerals bearing niobium, titanium and barium. The flotation process as a separation and enrichment of the acceptable levels by the P₂O₅ market (35%) is the most efficient. Brazilian mineral industries use synthetic collectors from international companies. Many of these oils were initially produced to sort out the demand for biodiesel production, with their market being …

Effect Of Calcium Concentration On Calcite Flotation From Apatite Using Carbonic Gas, Amanda Soares, Suzanne Ferreira De Mello, Rafael Teixeira Rodrigues

Beneficiation of Phosphates VIII

The most widely industrial flotation process for the treatment of phosphate ores containing carbonate as gangue minerals, especially in China, Saudi Arabia, Morocco, use strong acids, such as H₃PO₄ and H₂SO₄ as apatite depressants, and the carbonate minerals (especially calcite and dolomite) are selectively floated with the use of fatty acids as collectors at pH between 5.5 - 6.0. The flotation process currently considered for apatite concentration for the Santa Quitéria ore (Brazil) was developed in the middle of the 1980´s at the Center for Development of Nuclear Technology and involves bulk flotation of …

History And Future Of Phosphate Mining And Beneficiation In South Africa, Marius Porteus

Beneficiation of Phosphates VIII

The major igneous phosphate deposit in Phalaborwa was discovered in 1904. The establishment of the Phosphate Development Corporation (Foskor) in 1951 catalyzed the development and growth of phosphate mining and phosphate fertilizer manufacturing in South Africa as well as associated businesses. This keynote address covers four major topics: 1) chemical and mineralogical properties of major phosphate deposits in South Africa, 2) mining and beneficiation practices, 3) technical challenges and evolution of processing technologies, and 4) future trends.

Mine Arnaud Project: Flotation Circuit Adjustment And Collector Reduction, Christine Croteau, Patrick Laflamme, Michel Lafontaine

Beneficiation of Phosphates VIII

The Mine Arnaud open deposit is located in Sept-Iles in the province of Quebec, Canada. The project, which is currently under development, consists of igneous apatite ore grading around 5% P₂O₅.The target metallurgical performances are set to a concentrate of P₂O₅ grade of at least 39% with an overall P₂O₅ recovery above 90%.The beneficiation of the ore is achieved by grinding to the liberation size, magnetic separation to remove the titaniferous magnetite and phosphate flotation. Most of Mine Arnaud’s apatite beneficiation flowsheet development testwork was realized by COREM. The most …

Hydrothermal Pre-Treatment Process Of Phosphogypsum For Enhanced Rare Earths Recovery, Volha Yahorava, Eugene Lakay, Wilma Clark

Beneficiation of Phosphates VIII

Phosphogypsum (PG) is an industrial waste of phosphoric acid (H₃PO₄) production from phosphate ore by sulphuric acid (H₂SO₄) digestion, commonly known as the “wet acid” process. Phosphate rock often contains some valuable rare earth elements (REE) and it is well-established that from 70% to 85% of REE originally present in the phosphate rock end up in the PG, with an average REE content of ~0.4 wt. % [1-4]. PG is considered to be an attractive secondary resource of REE. However, processes available currently for REE recovery are often very expensive, sometimes impractical, …