Gargi Seth

Metabolic flux analysis and pharmaceutical production.

Rational engineering of biological systems is an inherently complex process due to their evolved nature. Metabolic engineering emerged and developed over the past 20 years as a field in which methodologies for the rational engineering of biological systems is now being applied to specific industrial, medical, or scientific problems. Of considerable interest is the determination of metabolic fluxes within the cell itself, called metabolic flux analysis. This special issue and this review have a particular interest in the application of metabolic flux analysis for improving the pharmaceutical production process (for both small and large molecules). Though metabolic flux analysis has been somewhat limited in application towards pharmaceutical production, the overall goal is to: (1) have a better understanding of the organism and/or process in question, and (2) provide a rational basis to further engineer (on both metabolic and process scales) improved pharmaceutical production in these organisms. The focus of this review article is to present how experimental and computational methods of metabolic flux analysis have matured, mirroring the maturation of the metabolic engineering field itself, while highlighting some of the successful applications towards both small- and large-molecule pharmaceuticals.

Engineering cells for cell culture bioprocessing - Physiological fundamentals

In the past decade, we have witnessed a tremendous increase in the number of mammalian cell-derived therapeutic proteins with clinical applications. The success of making these life-saving biologics available to the public is partly due to engineering efforts to enhance process efficiency. To further improve productivity, much effort has been devoted to developing metabolically engineered producing cells, which possess characteristics favorable for large-scale bioprocessing. In this article we discuss the fundamental physiological basis for cell engineering. Different facets of cellular mechanisms, including metabolism, protein processing, and the balancing pathways of cell growth and apoptosis, contribute to the complex traits of favorable growth and production characteristics. We present our assessment of the current state of the art by surveying efforts that have already been undertaken in engineering cells for a more robust process. The concept of physiological homeostasis as a key determinant and its implications on cell engineering is emphasized. Integrating the physiological perspective with cell culture engineering will facilitate attainment of dream cells with superlative characteristics.

A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies

To meet increasing demands for efficient and streamlined production processes of therapeutic antibodies, improved methods of screening clones are required. In this article, we examined the potential of using antibody transcript levels as criteria for clone screening. We evaluated the QuantiGene Plex, a commercially available, high‐throughput assay for simultaneously measuring multiple transcripts from cell lysate. Using the development of stable Chinese hamster ovary cell lines as examples, we investigated the relationship between transcript and antibody levels through several rounds of screening. First, we observed that measured heavy chain transcript levels are generally correlated with specific productivity, enabling the identification of high‐producing clones from mRNA. Second, we observed that low ratios (<1.5) of light to heavy chain transcript levels may be indicative of high antibody aggregation levels, allowing for the rapid identification and elimination of clones of questionable product quality. Therefore, an efficient process of identifying high‐producing clones of desirable product quality is possible by using QuantiGene Plex assay to measure antibody transcript levels. Biotechnol. Bioeng. 2009;102: 1107–1118.

Development of a New Bioprocess Scheme Using Frozen Seed Train Intermediates to Initiate CHO Cell Culture Manufacturing Campaigns

Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi‐product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 106 cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Biotechnol. Bioeng. 2013; 110: 1376–1385.

Freezing mammalian cells for production of biopharmaceuticals.

Cryopreservation techniques utilize very low temperatures to preserve the structure and function of living cells. Various strategies have been developed for freezing mammalian cells of biological and medical significance. This paper highlights the importance and application of cryopreservation for recombinant mammalian cells used in the biopharmaceutical industry to produce high-value protein therapeutics. It is a primer that aims to give insight into the basic principles of cell freezing for the benefit of biopharmaceutical researchers with limited or no prior experience in cryobiology. For the more familiar researchers, key cell banking parameters such as the cell density and hold conditions have been reviewed to possibly help optimize their specific cell freezing protocols. It is important to understand the mechanisms underlying the freezing of complex and sensitive cellular entities as we implement best practices around the techniques and strategies used for cryopreservation.

Toward genomic cell culture engineering

Genomic and proteomic based global gene expression profiling has altered the landscape of biological research in the past few years. Its potential impact on cell culture bioprocessing has only begun to emanate, partly due to the lack of genomic sequence information for the most widely used industrial cells, Chinese hamster ovary (CHO) cells. Transcriptome and proteome profiling work for species lacking extensive genomic resources must rely on information for other related species or on data obtained from expressed sequence tag (EST) sequencing projects, for which burgeoning efforts have only recently begun. This article discusses the aspects of EST sequencing in those industrially important, genomic resources-poor cell lines, articulates some of the unique features in employing microarray in the study of cultured cells, and highlights the infrastructural needs in establishing a platform for genomics based cell culture research. Recent experience has revealed that generally, most changes in culture conditions only elicit a moderate level of alteration in gene expression. Nevertheless, by broadening the conventional scope of microarray analysis to consider estimated levels of transcript abundance, much physiological insight can be gained. Examples of the application of microarray in cell culture are discussed, and the utility of pattern identification and process diagnosis are highlighted. As genomic resources continue to expand, the power of genomic tools in cell culture processing research will be amply evident. The key to harnessing the immense benefit of these genomic resources resides in the development of physiological understanding from their application.

Stem cell culture engineering

Abstract Stem cells have the capacity for self renewal and undergo multilineage differentiation. Stem cells isolated from both blastocysts and adult tissues represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self‐renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, and expansion or survival of specific cell types are key in controlling the differentiation towards specific cell lineages. Our limited knowledge of their growth conditions as well as lack of appropriate markers associated with different stages of differentiation hinders the widespread use of stem cells. However, a variety of bioreactors exist for cell cultivation that can be readily adapted to provide a well controlled environment for studying the process of stem cell propagation and differentiation. Here we review the advances made in the field of stem cell culture; and discuss the employment of different platforms for stem cell cultivation that will facilitate the advancement of stem cell science into the realm of application based technology in the foreseeable future.