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 …

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 …

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 …

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 …

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 …

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 …

Integrated Strategies For The Production, Maturation And Storage Of Functional Cardiomyocytes Derived From Human Pluripotent Stem Cells, Claudia Correia, Paula Alves

Cell Culture Engineering XV

The production of cardiomyocytes (CM) from human pluripotent stem cells (hPSC) holds great promise for cardiotoxicity drug testing, disease modeling and cardiac regeneration [1]. However, the complex nets of signalling pathways involved in cardiomyogenesis compromises the effectiveness of the existing differentiation protocols to reproducibly produce high-quality CM from hPSC (hPSC-CM). Produced hPSC-CM are immature compared with adult CMs, express typical fetal cardiac genes, have immature electrophysiological properties and use glucose as major energy source [2]. The applicability of hPSC-CM in the clinic/industry is also dependent on the development of efficient methods for worldwide shipment of these cells. In this study …

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). …

Modelling Short-Latency Disparity-Vergence Eye Movements Under Dichoptic Unbalanced Stimulation, Agostino Gibaldi, Guido Maiello, Peter J. Bex, Silvio P. Sabatini

MODVIS Workshop

Vergence eye movements align the optical axes of our two eyes onto an object of interest, thus facilitating the binocular summation of the images projected onto the left and the right retinae into a single percept. Both the computational substrate and the functional behaviour of binocular vergence eye movements have been the topic of in depth investigation. Here, we attempt to bring together what is known about computation and function of vergence mechanism. To this aim, we evaluated of a biologically inspired model of horizontal and vertical vergence control, based on a network of V1 simple and complex cells. The …

Measuring And Modeling Shared Visual Attention, Jeffrey B. Mulligan, Patrick Gontar

MODVIS Workshop

Multi-person teams are sometimes responsible for critical tasks, such as flying an airliner. Here we present a method using gaze tracking data to assess shared visual attention, a term we use to describe the situation where team members are attending to a common set of elements in the environment. Gaze data are quantized with respect to a set of N areas of interest (AOIs); these are then used to construct a time series of N dimensional vectors, with each vector component representing one of the AOIs, all set to 0 except for the component corresponding to the currently fixated …

Challenges In The Use Of Scale-Down Models For Understanding And Mitigating Process Variations Of A Monoclonal Antibody Production Process, Peter Russo

Cell Culture Engineering XV

Scale-down models are commonly used to execute process characterization studies, as well as to screen raw materials and to conduct investigations in support of manufacturing operations. During the clinical manufacture of a monoclonal antibody, consistent product quality was maintained; however, large variations in bioreactor harvest titer (> 2X) were observed between lots. Root cause analysis of this variation did not establish a linkage between culture performance and manufacturing execution or deviations. Further, small scale satellite reactor performance displayed lesser variability; thereby eliminating seed train and raw materials as potential root causes. Small scale investigations, using multiple scale-down models, were not …

Rapid Development Of A Perfusion Process With High Productivity, Sen Xu, Linda Hoshan, Balrina Gupta, Hao Chen

Cell Culture Engineering XV

Continuous process improvement is required during a biologics process life cycle to increase process yield and reduce cost. In this poster, we will present a case study in late stage process development where everything was changed but the product quality. The following major changes were made when developing the new process:

• Production cell line – subclone of the original clone used.
• Media – from hydrolysate-containing feeds to fully chemically-defined feeds.
• Feeding strategy – from fixed volume/fixed schedule feeding to daily on-demand feeding.

Media components were optimized through spent media analysis and mechanistic understanding to accommodate the new feeding strategy. High-throughput …

Bioanalytical Comparability Of Biotechnology Products Subject To Changes In Their Manufacturing Process, Jose Menezes, Maria Campos, Pedro Felizardo

Cell Culture Engineering XV

Over lifecycle, from development through all industrialization stages and then over manufacturing history across multiple sites, a biotechnology product must show very high levels of quality consistency, as that is a pre-requisite for safety and efficacy to patients (cf. ICH Q5E, 2004).

Bioanalytical similarity has acquired over the last years another meaning with the introduction of biosimilars: products that in principle cannot be identical to their reference products as processes used and other design options are not exactly identical, leading for example to minor differences in clinically inactive components. However, biosimilars must be proven to be pharmacologically identical to their …

Real Time Prediction And Control Of Glycoform Profile Of Mammalian Cell Cultures Using In Silico Glycosylation Model Coupled With Extracellular Metabolites, Sha Sha, Seongkyu Yoon

Cell Culture Engineering XV

In silico mechanistic models for glycosylation are investigated for biotherapeutics development and production. The models systematically describe the transport-kinetics of glycosylation process and serve as a prediction framework for glycan profile. However, inputs for those models are generally limited to intracellular parameters, such as enzymatic activity and nucleotide sugar concentration. These intracellular metabolites are unfortunately inaccessible in process. To modulate those models for glycoform prediction in culture process, linkage between measurable culture variations and the intracellular metabolites change will be thoroughly investigated. The measurable metabolites and intracellular variables associated with in silico glycosylation model are integrated in the modeling. Despite …

Scalability Of The Mobius® Single-Use Bioreactor From Bench To Clinical Scale: Examination Of Key Engineering Parameters And Robustness, Lee Madrid

Cell Culture Engineering XV

To successfully implement a large scale single-use bioreactor platform, equivalent performance, i.e. scalability, with smaller sized bioreactors across the platform must be demonstrated. With the recent development of a single-use clinical scale (2000 L) bioreactor, the EMD Millipore bioreactor portfolio now spans from the bench-scale (3 L), through the small-scale (50 L) and pilot-scale (200 L) up to the manufacturing process volume range. The performance design space of the entire Mobius® Single-use Bioreactor platform was characterized using several key engineering parameters including oxygen mass transfer coefficient (kLa), power per unit volume, Reynolds number (Re), mixing time and tip speed. Based …

Overcoming Scale-Up Challenges With A Non-Robust Cell Line, Sigma Mostafa, Brian Baker, Abhinav Shukla

Cell Culture Engineering XV

Cell culture scale up success depends on robust cell line, process, and equipment. Mixing characteristics of bench scale through large scale bioreactors need to be well understood and a reliable scale up parameter needs to be used. The cell culture raw material lot to lot variability as well as hydration protocol reliability needs to be considered. The cell culture process parameter ranges need to be broad enough to account for system variability and the process should not run at the edge of failure. In our experience, greater than 95% of cell lines are robust and easily scalable. Cell line robustness …