Biomedical Engineering and Bioengineering Commons^™

Integration Of Bioprocess Design With Transcriptomic And Metabolomic Characterization For The Expansion Of Human Pluripotent Stem Cells, Marta Silva

Cell Culture Engineering XV

Human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC), have an enormous potential as source for cell replacement therapies. However, the clinical application of hPSC has been hindered by the lack of robust protocols able to sustain production of high cell numbers maintaining their phenotype. To facilitate the bioprocess optimization aiming at higher cell yields, the analysis of transcriptomic and metabolomic profiling would provide important insights about stem cell phenotype in different culture conditions. Within this context, we developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions using stirred-tank bioreactors. Moreover, we explored the …

Development Of An Adherent Cell Based Virus Production Process In Mobius® Singleuse Bioreactor, Michael Cunningham

Cell Culture Engineering XV

Cell culture production processes are being developed as an alternative to the egg-based influenza vaccine production. The egg based method is slow and is subject to avian influenza outbreaks which can leave the human population at risk. The cell culture derived method has several advantages over the egg-based production. It is a robust method which can produce a large volume of vaccine in a short period of time thereby reducing the risk of vaccine supply shortages. This presentation focuses on the development of an upstream MDCK cell based influenza A/WS/33 flu production process using Cytodex³ microcarriers in a 3L …

Production Of Stable, Immunogenic Foot-And-Mouth Disease Vaccine In A Chemicallydefined, Serum-Free Medium Optimized For Bhk-21 Cells, Paul Gulde

Cell Culture Engineering XV

BHK-21 cells are an important bioprocessing host for the production of recombinant proteins and vaccines. The largest application of these cells is the production of Foot-and-Mouth Disease (FMD) and Rabies vaccines for the animal health market. Commercial FMD vaccine production relies on classical cell culture media supplemented with bovine serum. This serum is sourced locally from FMD-vaccinated cattle and thus requires antibody precipitation with PEG to allow supplementation during cell culture scale-up. This step diminishes the nutritional value of sera and also increases variability. To reduce serum use during the virus production phase, the process depends on media supplementation with …

Transient Production Of Vlps In Hek 293 Cells And The Evaluation Of Parameters Influencing Transfection And Expression, Daniel Blackstock

Cell Culture Engineering XV

Transient transfections can serve as an attractive alternative to generating cell lines which are difficult to engineer, such as those producing virus-like particles (VLPs). Transient methods can also allow for the production of large quantities of material within short periods of time. However, in order to make the process viable on a steady schedule or a larger scale, a broad realization of the potential variations of the process and the resulting effect must be explored. Upon identifying the impactful variations, the transient process can be properly controlled and become less challenging to carry out and reproduce.

In this work we …

Application Of 13c Flux Analysis To Identify High-Productivity Cho Metabolic Phenotypes, Jamey Young

Cell Culture Engineering XV

Industrial bioprocesses place high demands on the intermediary metabolism of host cells to meet the biosynthetic requirements for maximal growth and protein expression. Identifying host cell metabolic phenotypes that promote high recombinant protein titer is a major goal of the biotech industry. ¹³C metabolic flux analysis (MFA) provides a rigorous approach to quantify these metabolic phenotypes by applying isotope tracers to map the flow of carbon through intracellular metabolic pathways. We have conducted a series of ¹³C MFA studies to examine the metabolic impacts of recombinant IgG expression using two common CHO expression systems, the glutamine synthetase (GS) …

Understanding And Controlling Sialyation In A Cho Fusion Protein At Lab And Manufacturing Scale Using Targeted Omics Techniques, Amanda Lewis, Nelly Aranibar, William Croughan, Nicholas Abu-Absi, Bethanne Warrack, Michael Borys, Michael Reily, Zheng Jian Li

Cell Culture Engineering XV

Biologics, including antibodies, hormones and cytokines, represent an increasingly important class of therapeutics, with 7 of the 10 top selling drugs from 2013 in this class. The glycosylation distribution of these proteins is an important characteristic that can impact biological activity, circulatory half-life, and immunogenicity. One property that affects glycoproteins is the terminal addition of N-acetylneuraminic acid (sialic acid) to glycosylation chains. Despite the importance of glycosylation in many therapeutic proteins, limited information is available to date linking process parameters to changes in glycosylation distribution. The majority of the work that has been done is limited to a small number …

Mammalian Systems Biotechnology: An Integrative Framework For Combining In Silico Modeling And Multi-Omics Datasets In Different Cho Parental Cell Lines, Dong Yup Lee, Sarantos Kyriakopoulos, Meiyappan Lakshmanan, Kok Siong Ang, Alison Lee, Xuezhi Bi, Andy Tan, Ying Swan Ho, Peiqing Zhang, Yuansheng Yang, Say Kong Ng

Cell Culture Engineering XV

The increasing availability of multi-omics data from Chinese hamster ovary (CHO) cell cultures entails both opportunity and challenges toward next generation cell culture engineering. Herein, we present a comprehensive and integrative framework to systematically combine trancriptome, proteome, metabolome and glycome datasets in conjunction with a genome-scale metabolic model of CHO cells. We then apply the framework to compare and contrast the metabolic characteristics of the three commonly used parental cell lines (CHO-K1, CHO-DUKXB11 and CHO-DG44) so that “global” attributes of the parental hosts (e.g. growth related characteristics, glycosylation patterns, etc.) could be highlighted (Figure 1).

The unique characteristics of the …

A Quality By Design (Qbd) Approach To Cell Culture Process Characterization, Melissa Mun

Cell Culture Engineering XV

Quality by Design (QbD) is a science- and risk-based approach to development that begins with predefined objectives and emphasizes product and process understanding as well as process control. This case study will describe a QbD approach to characterizing a monoclonal antibody (MAb) cell culture process. Process parameters to study were selected through a risk ranking and filtering assessment based on historical development data, manufacturing history, and scientific understanding. Statistically designed, multifactor process characterization studies were performed in scale-down bioreactors to build understanding of parameter impacts on critical quality attributes (CQAs) and key performance indicators (KPIs). Production culture process characterization studies, …

Biological Relevance Of Yap Regulation By Wnt Signaling During Neural Tissue Patterning Of Human Induced Pluripotent Stem Cells, Yan Li

Cell Culture Engineering XV

Human induced pluripotent stem cells (hiPSCs) have special ability to self-assemble into neural microtissues or mini-organ like structures (e.g., mini-brains). In this process, Wnt signaling impacts regional patterning and positional identity of hiPSC-derived neural progenitors. One important function of Wnt signaling is to regulate Yes-associated protein (YAP) expression (nuclear or cytoplasmic), the pivotal regulator of cell proliferation and differentiation during organ growth and tissue generation. However, the crosstalk between Wnt and YAP expression during neural differentiation of hiPSCs has not been well investigated. The objective of this study is to reveal the capability of Wnt signaling in the regulation of …

Industrialization Of Adenoviral Vector Production In Fixed Bed Bioreactor And Amplification Of Primary Liver Cells In Xpansion® Bioreactor: Autologous Insulin Producing Cells For The Treatment Of Diabetes, From Bench To Clinical Scale, Rachel Legmann

Cell Culture Engineering XV

Diabetes is a major global health problem with over 370 million diabetics and an estimated 550 million by 2030. Current therapies rely on recombinant insulin injection to the patients several times a day to control glucose level but do not address the fundamental problem; the loss of insulin producing cells of the pancreas. Orgenesis developed a cell therapy to replace these cells by taking a small biopsy from a patient’s liver, growing the cells in flatware treating these cells with adenovirus vectors containing the genes required to transdifferentiate them to insulin producing cells. This approach allows the diabetic patient to …

Effect Of Cell-Surface Interactions On Monocyte-Derived Immunotherapy Products, Corinne Hoesli

Cell Culture Engineering XV

Monocyte-based cellular therapy products have been approved by the FDA for immunotherapy applications. The transition from a research and preclinical setting to the clinic is typically associated with a shift open culture systems such as polystyrene flasks to closed cell culture systems. Closed systems such as fluoropolymer-based bags offer benefits from a handling and safety perspective. However, the interactions between cells and fluoropolymer surfaces may differ from polystyrene surfaces. Differences between the physicochemical properties of the materials may profoundly impact the type and conformations of the proteins adsorbing to the surfaces. In turn, differences in the protein adlayer formed on …

Non-Invasive Real-Time Monitoring Of Glucose And Lactate By Nir-Spectroscopy During Perfusion Cho Culture, Jean Hamel, Anita Wamakima, Ruifan Pei

Cell Culture Engineering XV

Near-infrared spectroscopy (NIR) has been used to monitor glycerol and methanol, non-invasively during cultures of Pichia pastoris^{1, 2}. In this work, glucose and lactate were measured in Chinese Hamster Ovary (CHO) perfusion culture, in real time, using an online advanced NIR monitor and a reference offline biochemical analyzer. The 1.8-L culture started in the batch phase at 4 g/L glucose with 0.3 x 10⁶ cells/mL and reached 1.5 x 10⁶ cells/mL after 90 hours. Perfusion was then initiated and conducted for 10 days at 0.7 vvd, using a spiral membrane-less microfluidic device³. The maximum …

Development Of Chemically-Defined, Animal Component-Free Medium For Suspension Mdck Cell-Based Influenza Vaccine Production, Jenny Bang

Cell Culture Engineering XV

Cell culture-based vaccine production is advantageous over chicken eggs-based production because it does not rely on the availability of eggs and can be performed on a larger scale in a timely manner. Madin-Darby canine kidney (MDCK) cells are favorably used for vaccine production, particularly for influenza vaccines. However, there are several drawbacks to the traditional method of culturing MDCK cells. MDCK cells require undefined supplement for culturing, such as serum or hydrolysates which pose regulatory concerns especially when making pharmaceutical products for human use. Furthermore, MDCK cells grow in adherent culture which requires significant surface area or the use of …

Neural Patterning Of Human Induced Pluripotent Stem Cells For Studying Neurotoxicity, Yan Li, Yuanwei Yan

Cell Culture Engineering XV

Existing models using adult human neural stem cells have the restricted access. Human induced pluripotent stem cells (hiPSCs) can generate allogeneic or patient-specific neural cells/tissues and even mini-brains to provide robust in vitro models for applications in drug discovery, neurological disease modeling, and cell therapy. Toward this goal, the objective of this study is to construct 3-D neural models from hiPSCs through the scalable embryoid body-based suspension culture which can generate cortical glutamatergic neurons and motor neurons by tuning the sonic hedgehog (SHH) signaling. The differentiation of human iPSK3 cells was induced using dual inhibition of SMAD signaling with LDN193189 …

Genomics Based Methodology Of Cell-Culturemedia Formulation For Improved Biotherapeutic Productivity And Quality Consistency, Hemlata Bhatia, Seongkyu Yoon

Cell Culture Engineering XV

Biosimilar drugs are emerging very fast as the patents of innovator’s drugs are expiring. Media formulation development has to be carried out for each biosimilar, which takes up a significant amount of time and generally, a random approach is taken to meet comparability requirement and improve the product titer. A targeted approach with genome information of host cell as a function of media composition can provide useful information to explain the product titer variability and comparability. A 2-step correlation model explaining relationship between cell-culture productivity and quality, and media compositions taking gene expression as intermediate attributes can provide a precise …

Towards Integrated Continuous Viral Vaccines Production Using Two-Stage Bioreactor Systems, Felipe Tapia

Cell Culture Engineering XV

Typically, many cell culture-derived viral vaccines are being produced in batch processes. However, with increasing demands of a growing world population for potent, safe and affordable vaccines, manufacturing technologies need to be further optimized. In particular, the establishment of two-stage bioreactors appears as an interesting option for continuous production of viral vaccines. In these systems cell growth and virus propagation are being performed in separated vessels [1], which make them suitable for production of lytic viruses, such as influenza and Modified Vaccinia Ankara virus (MVA). MVA virus has received much clinical attention, because it is as a promising vector candidate …

Evaluation Of Public Genome References For Rna-Seq Data Analysis In Chinese Hamster Ovary Cells, Huong Le, Chun Chen, Chetan Goudar

Cell Culture Engineering XV

Recent advances in next-generation sequencing technologies have led to the emergence of RNA-Seq as the preferred transcriptomic tool in the biopharmaceutical industry. However, an important challenge with deploying RNA-Seq to characterize CHO cells is the absence of a common genomic reference for this species. In most published CHO cell transcriptomic studies, RNA-Seq reads are assembled into de novo genomic references which were subsequently used for mapping of the constituent reads. Such an approach makes it difficult to compare results across studies due to the incomplete and non-universal nature of those assemblies. To address this challenge, we evaluated two publicly available …

Elucidating Cell Line And Tissue Differences Derived From Cricetulus Griseus By Transcriptomics And Proteomics, Kelley Heffner

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the dominant host for recombinant protein production. This project aims to expand our knowledge by conducting transcriptomics and proteomics experiments on CHO cell lines and Chinese hamster tissues in order to obtain a larger set of genetic engineering targets for improving bioprocess development. This study will increase data sets using cell line comparisons and investigate how mRNA and protein levels change from Chinese hamster lineage. Quantitative ‘omics data from CHO cell lines and Chinese hamster tissues data was obtained by RNAseq (transcriptome) and labeled tandem mass tag mass spectrometry (proteome). …

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 …

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 …

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 …

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. …

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 …

An Innovative Protein Delivery System For Therapeutic Cells, Jean Pascal Lepetit

Cell Culture Engineering XV

Cell therapy has been on the rise in recent years. It consists in the treatment of therapeutic cells to be transferred to human patients, and is applied in multiple pathologies such as hemophilia or muscular dystrophies. Several cell treatment solutions are currently developed, including the use of transcription factors or genome editing technologies. These ex vivo technologies often require gene transfer to express the therapeutic protein in the treated cells. The genes are delivered mainly by transfection or viral transduction, causing regulatory limitations for clinical studies.

A promising strategy is to avoid the use of nucleic acids by direct delivery …

Agitation Strategies For The Culture And Detachment Of Human Mesenchymal Stem Cells (Hmscs) From Microcarriers In Multiple Bioreactor Platforms, Alvin Nienow

Cell Culture Engineering XV

Unlike cell culture for biopharmaceuticals, where the product of interest is usually a recombinant protein, for regenerative medicine, the cells form the basis of the therapeutic. For the expansion of cells for allogeneic purposes, work, mainly in spinner flasks, has led to culture on microcarriers. For processes involving mass transfer and reaction with particles in stirred reactors (e.g., crystallization, catalytic reaction), it is essential that particles are at least just fully suspended, agitator speed N_JS, for effective mass transfer to and from the particles. Though gentle agitation has generally been recommended, it has not previously been defined in …

Lipidomics For Robust High Performance Process Development, Laetitia Malphettes, Gregory Mathy, Laura Gimenez

Cell Culture Engineering XV

As the biopharmaceutical industry reduces the risk of potential contaminations by adventitious agents and increases process yields, high performance cell culture processes have been developed that rely on animal-free peptide-free, protein-free chemically defined and lipid-free media. These processes rely on cell lines that have been adapted to these lipid-free production conditions and have developed very effective lipid production capacities. These lipid-production capacities result in new challenges in the harvest and purification steps such as filterability, ability to clean resins and resin reuse. This oral presentation will show case how lipidomics can provide insights and opportunities to control the interactions between …

Development Of A Process Analytical Technology (Pat) Infrastructure For Future Biologics Upstream Processing, Mathieu Steefland

Cell Culture Engineering XV

Process Analytical Technology (PAT) involves the timely analysis of Critical Quality Attributes (CQAs) and Critical Process Parameters (CPPs) with the aim to improve the process control strategy resulting in a better process performance and robust product quality. The progress of PAT implementation in upstream bioprocessing has been slow mainly due to the complexity of the process steps with many parameters involved, and the complexity of the product and the culture medium. While the first approved PAT-based Real Time Release application in the small molecules field already celebrating its 10th anniversary in 2016, PAT for upstream bioprocessing is only now maturing. …

Cell-Derived Microparticles For Cell Therapy, Cargo Delivery, And Applications In Cho-Cell Biotechnology, Terry Papoutsakis, Jinlin Jiang, Chen-Yuan Kao

Cell Culture Engineering XV

Mammalian cells release into the extracellular environment microparticles (MPs; less than 1 micron) under some stress or activation process. MPs result from direct budding off the plasma membrane, and are important in intercellular communication by transferring RNA, proteins, and lipids between cells. Cells endow their MPs with signaling or functional molecules, to target specific cell types. They enrich their MPs in specific miRNAs, piRNAs, and long ncRNAs to program or reprogram target cells towards functional differentiation or specific cellular actions. Cells also use MPs to get rid of “death molecules”, and/or promote cell survival and “renewal” of target cells. We …

Intracellular Characterization Of Gag-Gfp Vlp Production Upon Peimediated Transient Transfection Of Hek 293 Cells, Laura Cervera, Irene Gonzalez Dominguez, Mercedes Segura, Francesc Godia

Cell Culture Engineering XV

Transient Gene Expression is a fast, flexible, and cost-effective approach to produce high-quality products that circumvents the time and cost required for the generation of stably transfected cell lines. However, the levels of recombinant protein produced by TGE, tend to be significantly lower than those of stable cell lines. Despite the continued interest in transient gene expression approaches, little is known about the transfection process at intracellular level, particularly for complex products such as VLPs. The kinetics of PEI-mediated transient transfection was studied with the aim of characterizing and understanding the complete process leading to VLP generation, and identifying important …

Modeling Perfusion For Medium Component Optimization Using Ambr15tm, Deila Lyons

Cell Culture Engineering XV

An increasing number of biopharmaceutical companies are employing intensified perfusion as a means to increase volumetric productivity. Traditional perfusion systems have successfully used media developed for fed-batch cultures when target cell densities are relatively low (e.g., 20x10⁶vc/mL) and perfusion rates are relatively high (e.g., 5 vvd). High perfusion rates can be desirable when producing labile proteins in order to reduce the effective retention time of the protein in the bioreactor. Commonly, intensified perfusion processes applied in the production of stable proteins reach cell densities greater than 50x10⁶vc/mL with perfusion rates below 2vvd. In intensified perfusion, the …