Are we prepared for animal cell technology in the 21st century?

Large scale animal cell culture for the production of complex therapeutic proteins has been a major success of the biotechnology industry. Today, approximately half of the $ 5 billion annual turnover of the biotechnology industry is based upon this technology, in many cases with reactors of more than 10 m³. As we look towards the 21 st century, however, we can see novel approaches to the production of therapeutic proteins, by means of gene and cellular therapies. These technologies present new engineering challenges to the animal cell technologist. Are we prepared to meet these challenges? The needs include: small-scale reactors for the preparation of autologous cell lines, methods for the production of viruses to be used as vectors in gene therapy, artificial organ and the processing of xenogenic cell lines and tissues for cellular implants in humans. More attention should be given to three-dimensional cell cultures. Mass transfer considerations need to be extended beyond just oxygen transfer, to include cellular communication in small systems; this is becoming increasingly important for the control and optimise growth and product formation. Apart from improvements of large-scale systems, substantial advantages could be gained by studying new methods for the production and delivery of therapeutic proteins, using small-scale cell culture systems. We should adapt teaching, regulatory, patent and clinical infrastructure to meet this challenge in a harmonious way.     

Applications of immortalized cells in basic and clinical neurology

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.     

CHO细胞表达糖蛋白的研究进展*

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)表达系统因具有较高密度培养、高表达和相对完整的蛋白质糖基化修饰系统等特点,成为生产糖蛋白广泛应用的宿主表达细胞之一。目前已产生不同的CHO细胞系和各种功能细胞株以满足对糖蛋白的大量生产和其他实验需求。近年来,随着基因工程、蛋白质工程、细胞工程和发酵调控等技术的发展应用,由CHO细胞生产糖蛋白的产量和糖基化修饰程度取得了突破。然而,随着生物制品市场对于糖蛋白的需求增加,如何获得大量、均质的糖蛋白也成为急需解决的问题。综述了不同工程CHO表达系统的研究、应用、糖基化修饰系统,以及影响外源糖蛋白在CHO系统表达和糖基化修饰的理化因素,结合文献总结并预测了未来CHO细胞表达系统研究的四个具有重大意义的研究方向,以期在未来可以改善由CHO细胞表达糖蛋白的产量和质量。     

Genomics and proteomics in process development: opportunities and challenges

Global gene expression profiling by genomic and proteomic analyses has changed the face of drug discovery and biological research in the past few years. The benefit of these technologies in the area of process development for recombinant protein production has been increasingly realized. This review discusses the application of genome-wide expression profiling tools in the design and optimization of bioprocesses, with the emphasis on the effect on process development of mammalian cell culture. Despite the lack of genome sequence information for most of the relevant mammalian cell lines used, these technologies can be applied during various process development steps. Although there are only a few examples in the literature that present a major improvement in productivity based on genomics and proteomics, further advances in analytical tools and genome sequencing technologies will greatly increase our knowledge at the molecular level and will drive the design of future bioprocesses.     

转基因雄性不育系及其在杂种种子生产上的应用

雄性不育为农作物杂种优势的利用开辟了一条经济有效的途径。本综述了利用生物技术培育转基因雄性不育的多种策略,以及繁育用作大田配制杂交种的母本雄性不育系的新方法;探讨了其应用于商业化杂种生产的重要性及前景。     

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.     

Inducible product gene expression technology tailored to bioprocess engineering

Bioprocess engineering has developed as a discipline to design optimal culture conditions and bioreactor operation protocols for production cell lines engineered for constitutive expression of desired protein pharmaceuticals. With the advent of heterologous gene regulation systems it has become possible to fine-tune expression of difficult-to-produce protein pharmaceuticals to optimal levels and to conditionally engineer cell metabolism for the best production performance. However, most of the small-molecules used to trigger expression of product or metabolic engineering product genes are incompatible with downstream processing regulations or process economics. Recent progress in product gene control design has resulted in the development of bioprocess-compatible regulation systems, which are responsive to physical parameters such as temperature or physiologic trigger molecules that are either an inherent part of host cell metabolism or intrinsic components of licensed protein-free cell culture media, such as redox status, vitamin H and gaseous acetaldehyde. While all of these systems have been shown to fine-tune product gene expression independent of the host cell metabolism some of them can be plugged into metabolic networks to capture critical physiologic parameters and convert them into an optimal production response. Assembly of individual product gene control modalities into synthetic networks has recently enabled construction of autonomously regulated time-delay or cell density-sensitive gene circuits, which trigger population-wide induction of product gene expression at a predefined time or culture density. We provide a comprehensive overview on the latest developments in the design of bioprocess-compatible product gene control systems.     

Semliki forest virus-based expression for versatile use in receptor research

Semliki Forest virus (SFV) vectors have been generated for highly efficient studies on gene expression in a variety of mammalian host cells, including immortalized cell lines as well as primary cells in culture. Moreover, SFV expression has been scaled up for mammalian suspension cultures in spinner flasks and bioreactors for production of large quantities of recombinant proteins for drug screening and purification. The strong preference of expression in neuronal cells in primary cell cultures, in organotypic hippocampal slices and in vivo has made SFV vectors attractive for neurobiological studies. Additionally, the engineering of novel, less cytotoxic and temperature-sensitive SFV mutant vectors has further increased their application range.     

克服位置效应提高外源基因在CHO细胞中稳定高效表达的策略

能够生产有功用的治疗性蛋白的一个重要前提是获得稳定的重组蛋白高表达细胞株,然而筛选一个能够持续稳定表达外源蛋白的重组细胞株是费时费力的过程。有多篇文献报道了重组蛋白细胞株表达的不稳定性。位置效应是高表达细胞株不稳定性的重要因素,克服或利用位置效应是当前获得稳定高表达重组蛋白细胞株的有效途径。为解决外源基因插入的随机性所带来的不可预知的后果,可以事先在CHO细胞基因组中筛选转录热点区域,再通过位点特异性或同源重组的方式,实现外源基因的定点整合。各种调节位置效应的DNA元件陆续被发现,可以利用它们去调控基因表达及增加细胞株的稳定性。     

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl‐2 family proteins such as Bcl‐2 and Bcl‐x_L potently inhibit apoptosis, no studies have examined the use of the Bcl‐2 family protein, Mcl‐1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl‐1 protein and a Mcl‐1 mutant protein that is not degraded by the proteasome in a serum‐free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl‐1 led to increased viabilities in fed‐batch culture, with cell lines expressing the Mcl‐1 mutant maintaining ～90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl‐1‐expressing cell lines were isolated that consistently showed increases in antibody production of 20–35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl‐1‐expressing cell lines, and Mcl‐1‐expressing cells exhibited 3‐fold lower caspase‐3 activation when compared with the control cell lines. Altogether, the expression of Mcl‐1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration

The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.     

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.     

Genetic optimization of recombinant glycoprotein production by mammalian cells

Genetically modified mammalian cells are the preferred system for the production of recombinant therapeutic glycoproteins. Other applications include engineering of cell lines for drug screening and cell-based therapies, and the construction of recombinant viruses for gene therapy. This article highlights contemporary core genetic technologies and emerging strategies for genetically engineering mammalian cells for optimal recombinant-protein expression.     

SEMLIKI FOREST VIRUS-BASED EXPRESSION FOR VERSATILE USE IN RECEPTOR RESEARCH

ABSTRACT

Semliki Forest virus (SFV) vectors have been generated for highly efficient studies on gene expression in a variety of mammalian host cells, including immortalized cell lines as well as primary cells in culture. Moreover, SFV expression has been scaled up for mammalian suspension cultures in spinner flasks and bioreactors for production of large quantities of recombinant proteins for drug screening and purification. The strong preference of expression in neuronal cells in primary cell cultures, in organotypic hippocampal slices and in vivo has made SFV vectors attractive for neurobiological studies. Additionally, the engineering of novel, less cytotoxic and temperature-sensitive SFV mutant vectors has further increased their application range.     

Human cell lines for biopharmaceutical manufacturing: history,status, and future perspectives

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.     

Antibody production by molecular farming in plants

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality.     

‘Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. ‘Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis‐related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.     

Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells

Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over‐expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess‐relevant characteristics of Chinese hamster ovary (CHO) cell‐derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell‐cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub‐optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR‐transgenic CHO‐derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four‐fold increase compared to the parental production cell line. mTOR‐based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Biotechnol. Bioeng. 2011; 108:853–866. © 2010 Wiley Periodicals, Inc.