Engineering Commons^™

Human Cardiac Stem Cells For Allogeneic Cell Therapies: Integrating Bioprocess Development And ‘Omics Characterization Tools, Patricia Gomes

Cell Culture Engineering XV

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Myocardial infarction (AMI) severely affects patients’ heart muscle and microvasculature, critically decreasing the number of functional cardiomyocytes (CM). Stem cell and protein based therapies became promising cardiac repair strategies since it was found that under pathological stress, resident cardiac stem cells (CSC) of the adult myocardium are activated by growth factors (GF) secreted by the surviving CM. Consequently, an auto/paracrine loop is triggered to maintain GF production, which enhances CSC activation and differentiation into new CM, endothelial and smooth muscle cells contributing to the repair of damaged myocardium [1]. Since …

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. …

Utilizing Rna-Seq Technique To Improve Molecular Understanding Of Chinese Hamster Ovary (Cho) Cell Bioprocessing, Yogender Kumar, Christpher Saski, Sarah Harcum

Cell Culture Engineering XV

Chinese Hamster Ovary (CHO) cells are an important biopharmaceutical cell line, accounting for the production of over 70% of the approved protein therapeutics. However several limitations exist with the use of CHO cell lines including low product titers. Understanding of CHO cells in bioprocessing has up until now relied heavily on empirical results with a limited knowledge of the intracellular dynamics. With the recent establishment of both Chinese hamster and CHO-K1 cell line genome assemblies, it is now possible to leverage the genomic resources to better understand and further improve CHO cell bioprocessing. In this study, RNA-Seq, next-generation transcriptome sequencing, …

Scale-Down And Initial Characterization Studies Of An Allogeneic Cell Therapy Manufacturing Process, Thomas Brieva

Cell Culture Engineering XV

Cancer therapies leveraging the immune system have been improving the course of disease. T-cell therapies are one of the modalities of immune therapy, which also include small molecules, proteins, and various types of immune cells. T-cell therapies include T-cell Receptor cells (TCR), Chimeric Antigen Receptor (CAR) T-cells, and Cytotoxic T-Lymphocytes (CTL). Although the nascent cell therapy industry faces many manufacturing challenges, it benefits from the vast experience of the blood industry and cell-produced biologics industry.

A notable manufacturing challenge for most T-cell therapies results from their autologous nature, in which cells from a patient are used to produce the therapeutic …

Multi-Omic Profiling Of Epo Producing Cho Cell Panel Reveals Metabolic Adaptation To Heterologous Protein Production, Daniel Ley

Cell Culture Engineering XV

The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed a multi-­omic physiological characterization including metabolic footprinting of amino acids, …

Michael Borys (Bristol- Myers Squibb) Incorporation Of Qbd Elements Into The Development And Characterization Of A Second Generation Process, Michael Borys, Nicholas Abu-Absi

Cell Culture Engineering XV

QbD principles are readily incorporated into mammalian cell processes to streamline process development and characterization. A key enabler of the implementation of these principles has been widespread adoption of platform technologies by the industry. This allows easy and efficient navigation of the QbD roadmap laid out in the A-Mab case study over the course of the development lifecycle of a product.

Here we examine the case of a 2nd generation process for a legacy product that was originally developed and approved using the traditional approach to process development and characterization. The goal of the 2nd generation process was to achieve …

Manipulation And Exploitation Of Micrornas For Enhanced Recombinant Protein Production In Cho Cells, Tulshi Patel, Lyne Josse, Mark Smales, Robert Young

Cell Culture Engineering XV

MicroRNAs (miRNAs) are regulatory non-coding RNA molecules around 22 nucleotides in length which control gene expression via translational repression of their cognate messenger RNA(s) (mRNAs). Regulating as much as 30% of the genome, miRNAs exhibit a high level of promiscuity as a single miRNA is able to target multiple mRNA targets and a single mRNA transcript can be under the regulation of several miRNAs. Thus, global regulation and control of almost all cellular processes and pathways is heavily dependent on the level and activity of miRNAs. This work has set out to investigate the biology of miRNAs in the industrially …

Development Of Plate-Based Sialic Acid Assays To Support Clone Screening And Early Stage Upstream Process Development, Julie Gardin, Yvette Tang

Cell Culture Engineering XV

Sialic acid, a post-translational modification, is an important product quality attribute of glycoproteins. The levels of sialic acid may impact solubility of the final product and the half-life (product clearance). In addition, the N-glycolylneuraminic acid (NGNA) form of sialic acid may elicit an immunogenic response in humans. In an effort to have a more high-throughput evaluation of these product quality attributes, two plate based assays were developed.

Specifically, a direct NGNA ELISA was developed using a polyclonal chicken anti-NGNA antibody. This assay was implemented as one of the clone screening tools to eliminate clones that may produce more of the …

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 …

Fluorescent Influenza-Like Particles And Control Over Their Composition, Marc Aucoin

Cell Culture Engineering XV

The Baculovirus Expression Vector System (BEVS) is a proven platform for making biologics. Despite being widely used, the system has typically relied on ‘brute force’ high expression. Building off of the handful of scientific publications that have shown that alternate promoter usage can promote higher functional protein yields, we have sought to generate baculovirus constructs that make use of a greater variety of promoters and genetic elements (shRNA/CRISPRi) to better control gene expression.

Materials and Methods

Genes for green fluorescent protein (GFP), DsRed2, and iRFP682, alone or as fusions to the human influenza A/PR/8/34 hemagglutinin (HA), matrix (M1) and neuraminidase …

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 …

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 …

Understanding And Overcoming Process Insults Through Application Of ‘Omics Technologies, Alan Gilbert, Kyle Mcelearney, Smitha Krishnan, Nicholas Alden, William Yang, Kyongbum Lee, Rashmi Kshirsagar

Cell Culture Engineering XV

Modern industrial process development, at both small and large corporations, usually consists of applying a well-characterized and established cell culture platform. Despite the high productivity available from these process platforms, difficult challenges remain, including with respect to the ability of the process to endure insults or disruptions. We previously demonstrated that overfeeding resulted in an undesirable increase in lactate production late in fed batch culture, which decreased productivity[i]. Here we report on metabolic flux analysis performed utilizing this process and isotopically labeling with multiple tracers (glucose and glutamate) delivered at five distinct time points of the cell culture …

Process Intensification Through Integration Of Upstream Perfusion Cell Culture With Downstream Continuous Chromatography In Monoclonal Antibody Production, Andreas Castan, Thomas Falkman, Eric Faldt, Teres Persson, Lisa Blomqvist, Annika Forss

Cell Culture Engineering XV

Process intensification is gaining interest as a strategy to reduce production costs, while improving product quality and throughput in the manufacturing of biopharmaceuticals. For a competitive production process, continuous or semi-continuous upstream and downstream processing can be employed. Compared with a process performed in batch runs, continuous processing allows for increased capacity utilization and eliminates or minimized the need for intermediate hold-up steps. Here, we describe the integration of a high-performing upstream cell culture process with downstream purification utilizing new emerging technologies such as periodic counter-current (PCC) chromatography and straight-through processing (STP).

A high-cell density perfusion process based on commercially …

Small-Scale Comparison Of Pseudoperfusion Feeding Strategies Using Basal And Concentrated Feed Media, Iona Bettinardi, Leda Castiho

Cell Culture Engineering XV

Perfusion has long been the industrial choice for the production of unstable proteins, and nowadays is being intensively studied also for stable proteins due to its ability to keep high viable cell densities and high volumetric productivities over long operation times. However, conventional perfusion is fed with basal medium at high dilution (or perfusion) rates, causing a dilution of the protein of interest (POI) and generating large volumes of harvest to be processed in the purification steps.

In this work, small-scale pseudoperfusion experiments were performed to compare the conventional perfusion strategy using basal culture medium (TC-LECC, Xell AG) with a …

Toward Development Of Continuous Bioprocesses: Comparison Of Fed-Batch And Perfusion Upstream Production Processes In Early Development, Jean Mclarty, Daryl Powers, Christine Hamel, Betsy Simons, Ken Karey

Cell Culture Engineering XV

Continuous Processing is an exciting development in the field of bioprocessing. The potential for quick response to market demands, decrease in infrastructure, increased flexibility and consistent product quality has resulted in a growing interest in Continuous Processing for production of all types of protein drugs (high or low volume, stable or unstable). Sanofi is developing a novel Integrated Continuous Manufacturing platform for biologics that utilizes an upstream perfusion process. While cell culture perfusion processes offer substantial benefits for commercial biologics production, implementation may present challenges in early development, where speed to first in man studies is critical. Here we present …

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 …}

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 …

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 …