Engineering Commons^™

Size Matters: Assessment Of A Larger Pore Hollow Fiber To Reduce Product Retention In Perfusion, Samantha Wang

Cell Culture Engineering XV

Traditionally, perfusion processes using either TFF or ATF technologies have utilized hollow fibers made from polymers such as polyethersulfone (PES) or polysulfone (PS) for cell retention. The pore sizes on these hollow fibers range from several hundred kD to a few µm. However, despite the relatively large size of the pores compared to that of the product, retention of product across the hollow fiber over time is a known and common problem in perfusion. Particle size analysis demonstrated accumulation of particles around 100nm in diameter, which are unable to pass through a typical 0.2µm PES membrane. As these particles are …

Process Robustness And Cell Line Variation In N-1 High Density Perfusion System, Haofan Peng

Cell Culture Engineering XV

In newly developed Biogen manufacturing processes, we are using perfusion technology to shift the cell growth phase into the seed train from the production reactor. This change improves facility throughput 30% by reducing time within the production bioreactor. In order to implement this approach, a hollow fiber based tangential flow filtration (TFF) system has been utilized in both lab and manufacturing settings. In defining the laboratory scale model, a case study showed that inadequate pumps and tubing can result in significant cell damage which was only observed at high cell densities. After integrating the non-spallation tubing with peristaltic pump, a …

Process Optimization For Semi-Continuous Virus Production At High Cell Densities, Daniel Vazquez

Cell Culture Engineering XV

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is still constrained by host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve a high-yield virus production processes. In this study, the development of a high-yield semi-continuous process for the production and purification of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza A/PR/8 in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented.

Methods. Depending on the required scale, high cell …

Technical Evaluation Of Rna-Seq And Microarray Approaches In Comparative Transcriptomics Analysis Of Cho Cells, Chun Chen, Huong Le, Chetan Goudar

Cell Culture Engineering XV

RNA-Seq has been replacing microarrays as the primary tool for comparative transcriptomics analysis. However, successful application of RNA-Seq to profile Chinese hamster ovary cells (CHO), the leading industrial cell line for recombinant protein production, was limited, primarily because of the inadequacy of genomic information for CHO cells. A compromised alternative to perform gene expression analysis and pathway or GO enrichment analysis in CHO cells was to map CHO genes to their mouse orthologs. Recent increased availability of CHO genomic references and the KEGG pathway reference for Chinese hamster has enabled direct gene expression analysis in the genome context of CHO …

Lipidomic Analysis To Enhance The Understanding Of Chinese Hamster Ovary Cells, Yue Zhang, Deniz Baycin, Michael Bowen, Michael Betenbaugh

Cell Culture Engineering XV

Chinese Hamster Ovary (CHO) cell lines are common hosts for the production of biotherapeutic proteins. Achieving high level of specific protein production by CHO cell lines remains a challenge. In order to address this issue, we are incorporating lipidomic analyses to study the role of lipids played in CHO-S cells.

In our study, we have applied chromatography (TLC) methods for lipid analysis in terms of lipid polarity. For polar lipids, 2-D HPTLC (2-dimensional high performance TLC) was used instead of conventional 1D- TLC by virtue of its high separation capacity. The eluting solvent system was optimized for the 1^{st …}

High Titer Transient Gene Expression Platform Based On Gs Cho Cell Line – Rapid Protein Expression Tool For Preclinical Drug Development, Yasha Rajendra, Gavin Barnard

Cell Culture Engineering XV

Most of the high yielding transient gene expression (TGE) methods for CHO cells reported in the literature involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. However, this is a laborious, time intensive process and also requires specific vector engineering for transient expression. Here, we present results from development of a high titer TGE process based on GS-CHO cells without resorting to host cell line engineering or TGE specific vector engineering. This was achieved by optimization of direct addition of DNA and PEI, use of DMA to enhance …

Improving Biologics Development By High Performance Glycoanalysis, Erdmann Rapp

Cell Culture Engineering XV

Glycomics is a rapidly emerging field that can be viewed as a complement to other „omics“ approaches including proteomics and genomics. Hence, there is a dramatic increase in the demand for analytical tools and specific databases in glycobiology, respectively, glycobiotechnology. In order to enhance and improve the comparatively small existing glycoanalytical toolbox, fully automated, highly sensitive, reliable, high-throughput and high-resolution analysis methods including automated data evaluation are required.

One very promising method is based on multiplexed capillary gelelectrophoresis with laser induced fluorescence detection (xCGE-LIF). The glycoanalytical approach established includes sample preparation and measuring methods, software, and database solutions to tackle …

Enhancing Site-Specific Cho Produced Antibody Through Media Optimization Using Metabolomics Approach, Ching Yang

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells have been widely used to produce recombinant protein. Several serum-free and chemically defined CHO medium are available for industrial manufacturing of recombinant antibodies. However, the effect on the cell metabolism and antibody productivity with different chemically defined basal media and feeds is still unclear. Recently, metabolic engineering become a powerful approach for medium optimization. In this study, we performed metabolic profiling (amino acids) by UPLC and compared two different commercial chemically defined basal media in batch and basal media with feeds in fed-batch culture system. We also compared the antibody productivity of site-specific high producing …

A Correction Method For Systematic Error In Metabolomic Time-Course Data, Stanislav Sokolenko, Marc Aucoin

Cell Culture Engineering XV

The growing ubiquity of metabolomic techniques has facilitated high frequency time-course data collection for many cell culture applications. Although the increasing resolution of metabolic profiles has potential to reveal important details about cell culture metabolism, more detailed results are subject to greater influence from measurement and data processing error. A number of common errors, stemming from metabolite extraction and internal standard addition, take the form of a dilution effect, where all observed concentrations feature a constant deviation relative to the true values. We have developed a simple technique to deal with such errors. A nonparametric smoothing fit was applied to …

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.

Site-Specific Glycan Analysis Of Proteins In Cell Culture Conditioned Media And Subcellular Fractions By Lc-Ms/Ms For Understanding The Impact Of Process Conditions On N-Glycosylation, Karina Bora De Oliveira, Nitin Agarwal, Christopher Barton

Cell Culture Engineering XV

Protein glycosylation, which involves the attachment of sugar residues to proteins, is an important post-translational modification that can influence the structure, pharmacological activity and stability of therapeutic proteins. The mechanisms by which cells modify and process these sugars therefore needs to be well understood and tightly controlled during protein production to ensure consistent product quality. Typically, glycosylation patterns are determined for the secreted therapeutic protein found in the conditioned media (CM). However, since glycosylation occurs through enzymatic reactions within the cellular endomembrane system, the glycosylation profile of the therapeutic protein in different intracellular compartments can also provide valuable insight into …

13c Flux Analysis In Industrial Cho Cell Culture Applications, Allison Mcafee, Jamey Young

Cell Culture Engineering XV

Industrial bioprocesses place extraordinary 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 stable 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 multiple stressors on CHO cell metabolism.

First, we analyzed the effects of …

Optimization Of A Defined Serum-Free Medium For The Production Of Therapeutic Human Myoblasts, Alain Garnier

Cell Culture Engineering XV

Duchenne Muscular Dystrophy (DMD) is a genetic disease affecting one boy out of 3500, which is due to a mutation in the dystrophin gene, inducing progressive and irreversible muscle degeneration. Cell therapy is the only means by which a DMD patient could recover part of his muscular mass and strength. We are presently collaborating with a team at the Quebec City University Hospital who is developing a cell therapy based on the graft to DMD patients of myoblasts obtained from biopsies on healthy and compatible donors. To do so, cells collected from donors need to be extensively multiplied. The standard …

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 …

Size Effect On The Magnetic Phase In Sr4Ru3O10, Yan Liu, Jiyong Yang, Weike Wang, Haifeng Du, Wei Ning, Langsheng Ling, Wei Tong, Zhe Qu, Zhaorong Yang, Minling Tian, Gang Cao, Yuheng Zhang

Center for Advanced Materials Faculty Publications

High quality Sr₄Ru₃O₁₀ nanoflakes are obtained by the scotch tape-based micro-mechanical exfoliation method. The metamagnetic transition temperature T_m^flake is found to decrease in line with the decrease of thickness, while the ferromagnetic (FM) phase, the ordinary, and anomalous Hall effects (OHE and AHE) are independent on the thickness of the flake. Analysis of the data demonstrates that the AHE reflects the FM nature of Sr₄Ru₃O₁₀, and the decrease of thickness favors the Ru moments aligned in the ab-plane, which induces a decrease of the metamagnetic transition …

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 …