Biomedical Engineering and Bioengineering Commons^™

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 …

Hek293 Suspension Cell Culture Platform For Production Of Viruses And Viral Vectors, Chun Shen, Sven Ansorge

Cell Culture Engineering XV

Viruses and viral vectors are extensively used as delivery systems for gene and cell therapies, oncotherapies and as vaccines or vectors for display or expression of antigens in vaccination strategies. Over many years, developments in cell culture technologies have been critical to enable mass production of viral vectors and have greatly contributed to facilitate pre-clinical and clinical trials for therapeutic applications. HEK293 is the most popular cell line for the production of adenoviruses (AdV), adeno-associated viruses (AAV), retroviruses and lentiviruses; and HEK293 cell suspension culture is the most efficient system for improved production of r-proteins and viral vectors by large-scale …