Metabolic engineering is rapidly developing, with a continuous stream of technological developments being employed to expand the portfolio of molecules produced in cell factories. For chemical production (e.g., amino acids, biofuels, among others), metabolic engineering has progressed through three phases [1]. Initially, biological products were obtained through random mutagenesis of production strains and large screening efforts. Improved microbial strains could be isolated, but mechanisms underlying the desired phenotype were often poorly understood [2]. Diverse molecular biology techniques facilitated the second phase, in which simple, intuitive modifications were made. The third phase now employs systems biology techniques to understand the effect of modifications on all other metabolic pathways and on cell physiology. Thus, we have entered an era in which metabolic engineering aims to improve microbial strains in a reproducible fashion, using complex designs based on detailed biochemical knowledge and computational model simulations. Here, we highlight the historical progression toward using systems biology in microbial metabolic engineering and compare this to the current status of mammalian production cell line development. Finally, we discuss the unique challenges in engineering mammalian cell lines for biotherapeutic production and outline how systems biology can facilitate metabolic engineering efforts for these platforms. The systems biology approach to metabolic engineering has been enabled by three primary advancements: whole-genome sequencing, gene editing tools and genome-scale models of cellular metabolism. The completion of the Escherichia coli K-12 genome sequencing effort in 1997 [3] provided a comprehensive parts list for targeted metabolic engineering and expanded the scope of our understanding of the machinery within this microbe. The further development of efficient genetic modification systems, such as the lambda Red recombination system [4], enabled the deployment of targeted metabolic engineering designs, such as the removal of competing pathways that divert flux away from the formation of a desired product. Predictions of the systemic effects of genetic modifications were enabled when the information in the sequenced genome was harnessed for the development of genome-scale models of metabolism [5]. These models contain all known biochemical reactions in a cell, thus allowing one to predict the overall impact of modifications on phenotypic traits such as growth rate and small molecule secretion. Systems biology approaches are now important tools in microbial metabolic engineering. Yim et al. genetically modified E. coli to produce 1,4-butanediol (BDO) by introducing heterologous genes to allow Hooman Hefzi Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
随着技术的不断发展，代谢工程正在迅速发展，以扩大细胞工厂生产的分子组合。对于化工生产(如氨基酸、生物燃料等)，代谢工程的发展经历了三个阶段。最初，生物制品是通过生产菌株的随机诱变和大量筛选工作获得的。改良的微生物菌株可以被分离出来，但是所期望的表型背后的机制往往知之甚少。不同的分子生物学技术促进了第二阶段，在这一阶段进行了简单、直观的修改。第三阶段现在采用系统生物学技术来了解修饰对所有其他代谢途径和细胞生理学的影响。因此，我们已经进入了一个代谢工程旨在以可复制的方式改善微生物菌株的时代，使用基于详细生化知识和计算模型模拟的复杂设计。在这里，我们强调了在微生物代谢工程中使用系统生物学的历史进展，并将其与哺乳动物生产细胞系发育的现状进行了比较。最后，我们讨论了用于生物治疗生产的工程哺乳动物细胞系的独特挑战，并概述了系统生物学如何促进这些平台的代谢工程工作。代谢工程的系统生物学方法已经通过三个主要进展实现:全基因组测序，基因编辑工具和细胞代谢的基因组尺度模型。1997年完成的大肠杆菌K-12基因组测序工作[3]为靶向代谢工程提供了一个全面的部件列表，并扩大了我们对这种微生物机制的理解范围。高效基因修饰系统的进一步发展，如lambda Red重组系统[4]，使得有针对性的代谢工程设计得以部署，例如去除将通量从所需产品的形成中转移的竞争途径。当测序基因组中的信息被用于开发代谢bb0的基因组尺度模型时，对遗传修饰的系统影响的预测成为可能。这些模型包含细胞中所有已知的生化反应，因此可以预测修饰对表型性状(如生长速度和小分子分泌)的总体影响。系统生物学方法现在是微生物代谢工程的重要工具。Yim等人通过引入异源基因对大肠杆菌进行转基因，使其产生1,4-丁二醇(BDO)，从而使Hooman Hefzi，加州大学圣地亚哥分校生物工程系，La Jolla, CA 92093, USA
{"title":"From random mutagenesis to systems biology in metabolic engineering of mammalian cells","authors":"Hooman Hefzi, N. Lewis","doi":"10.4155/PBP.14.36","DOIUrl":"https://doi.org/10.4155/PBP.14.36","url":null,"abstract":"Metabolic engineering is rapidly developing, with a continuous stream of technological developments being employed to expand the portfolio of molecules produced in cell factories. For chemical production (e.g., amino acids, biofuels, among others), metabolic engineering has progressed through three phases [1]. Initially, biological products were obtained through random mutagenesis of production strains and large screening efforts. Improved microbial strains could be isolated, but mechanisms underlying the desired phenotype were often poorly understood [2]. Diverse molecular biology techniques facilitated the second phase, in which simple, intuitive modifications were made. The third phase now employs systems biology techniques to understand the effect of modifications on all other metabolic pathways and on cell physiology. Thus, we have entered an era in which metabolic engineering aims to improve microbial strains in a reproducible fashion, using complex designs based on detailed biochemical knowledge and computational model simulations. Here, we highlight the historical progression toward using systems biology in microbial metabolic engineering and compare this to the current status of mammalian production cell line development. Finally, we discuss the unique challenges in engineering mammalian cell lines for biotherapeutic production and outline how systems biology can facilitate metabolic engineering efforts for these platforms. The systems biology approach to metabolic engineering has been enabled by three primary advancements: whole-genome sequencing, gene editing tools and genome-scale models of cellular metabolism. The completion of the Escherichia coli K-12 genome sequencing effort in 1997 [3] provided a comprehensive parts list for targeted metabolic engineering and expanded the scope of our understanding of the machinery within this microbe. The further development of efficient genetic modification systems, such as the lambda Red recombination system [4], enabled the deployment of targeted metabolic engineering designs, such as the removal of competing pathways that divert flux away from the formation of a desired product. Predictions of the systemic effects of genetic modifications were enabled when the information in the sequenced genome was harnessed for the development of genome-scale models of metabolism [5]. These models contain all known biochemical reactions in a cell, thus allowing one to predict the overall impact of modifications on phenotypic traits such as growth rate and small molecule secretion. Systems biology approaches are now important tools in microbial metabolic engineering. Yim et al. genetically modified E. coli to produce 1,4-butanediol (BDO) by introducing heterologous genes to allow Hooman Hefzi Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"355-358"},"PeriodicalIF":0.0,"publicationDate":"2014-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.36","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347917","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The past decade has seen significant instrumentation and methodological advances enabling much broader profiling of metabolites, proteins and nucleic acids. This in turn has been leveraged to enhance our sophistication and understanding of Chinese hamster ovary (CHO) cell metabolism in a bio processing environment. Of the three aforementioned fields, genomics has established itself as the preeminent technology platform for the foreseeable future largely due to a combination of throughput, comprehensive coverage and a relatively simple workflow. This has come to pass as a result of the tremendous advancements in nextgeneration sequencing (NGS) technology. These breakthroughs have lowered the barriers of cost and time associated with whole genome (DNA-Seq) and transcriptome (RNA-Seq) sequencing projects at an unprecedented rate, giving way to the ‘sequencing revolution’ [1]. The era of CHO genomics was ushered in by Xu and co-workers who applied NGS technology to create the first publically available CHO-K1 draft genome in 2011 [2]. CHO-K1 is but one of a handful of CHO host cell lines utilized by the bioprocessing industry. Indeed considering the extended time in culture and variety of adaptation strategies applied by the numerous labs working with CHO, every CHO host should be considered a unique cell line, irrespective of a shared common lineage [3]. Therefore, in subsequent publications by Lewis et al. [4] and Brinkrolf et al. [5] the CHO-K1 draft genome was expanded upon by sequencing the Chinese hamster genome. Having a Chinese hamster reference genome to facilitate the assembly of additional CHO genomes will benefit the community as a whole. However, the work is far from done as the quality of the genome is curtailed by gaps in sequencing coverage and incomplete gene annotations. A community of scientists working with CHOgenome.org is currently in the process of updating and correcting the CHO/hamster draft genomes, with particular interest in the sequencing gaps and annotations. This work will be critical to unlock the full benefits of having a high-quality genome to work with. Acknowledging the work that remains, the question becomes: how will NGS impact future bioprocess and what is the potential role of other ‘omics platforms?
{"title":"Next-generation bioprocess: an industry perspective of how the ‘omics era will affect future biotherapeutic development","authors":"Chapman Wright, S. Estes","doi":"10.4155/PBP.14.41","DOIUrl":"https://doi.org/10.4155/PBP.14.41","url":null,"abstract":"The past decade has seen significant instrumentation and methodological advances enabling much broader profiling of metabolites, proteins and nucleic acids. This in turn has been leveraged to enhance our sophistication and understanding of Chinese hamster ovary (CHO) cell metabolism in a bio processing environment. Of the three aforementioned fields, genomics has established itself as the preeminent technology platform for the foreseeable future largely due to a combination of throughput, comprehensive coverage and a relatively simple workflow. This has come to pass as a result of the tremendous advancements in nextgeneration sequencing (NGS) technology. These breakthroughs have lowered the barriers of cost and time associated with whole genome (DNA-Seq) and transcriptome (RNA-Seq) sequencing projects at an unprecedented rate, giving way to the ‘sequencing revolution’ [1]. The era of CHO genomics was ushered in by Xu and co-workers who applied NGS technology to create the first publically available CHO-K1 draft genome in 2011 [2]. CHO-K1 is but one of a handful of CHO host cell lines utilized by the bioprocessing industry. Indeed considering the extended time in culture and variety of adaptation strategies applied by the numerous labs working with CHO, every CHO host should be considered a unique cell line, irrespective of a shared common lineage [3]. Therefore, in subsequent publications by Lewis et al. [4] and Brinkrolf et al. [5] the CHO-K1 draft genome was expanded upon by sequencing the Chinese hamster genome. Having a Chinese hamster reference genome to facilitate the assembly of additional CHO genomes will benefit the community as a whole. However, the work is far from done as the quality of the genome is curtailed by gaps in sequencing coverage and incomplete gene annotations. A community of scientists working with CHOgenome.org is currently in the process of updating and correcting the CHO/hamster draft genomes, with particular interest in the sequencing gaps and annotations. This work will be critical to unlock the full benefits of having a high-quality genome to work with. Acknowledging the work that remains, the question becomes: how will NGS impact future bioprocess and what is the potential role of other ‘omics platforms?","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"371-375"},"PeriodicalIF":0.0,"publicationDate":"2014-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.41","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70348120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Is microfluidic perfusion culture the future for large-scale screening of human-induced pluripotent stem cells?","authors":"K. Hattori, S. Sugiura, T. Kanamori, K. Ohnuma","doi":"10.4155/PBP.14.25","DOIUrl":"https://doi.org/10.4155/PBP.14.25","url":null,"abstract":"","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"303-305"},"PeriodicalIF":0.0,"publicationDate":"2014-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.25","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fundamental to the efficient production of quality biopharmaceuticals is the selection, optimization and tailored manipulation of the mammalian cellular production host. Engineering of these cell factories, predominantly the Chinese hamster ovary cell and advancements in bioprocess regimens have led to greatly increased product titres. The ability of miRNAs to regulate gene expression on a global level has generated considerable interest in these molecules as potential cell engineering targets. In this review, we briefly describe their organization and biogenesis and discuss their attributes as engineering tools in Chinese hamster ovary cells. The development of particular engineering strategies based upon further dissection of miRNA behavior will be considered, with particular emphasis on encouraging examples in Chinese hamster ovary cells and their potential for further development.
{"title":"Bioprocess engineering: micromanaging Chinese hamster ovary cell phenotypes","authors":"Paul S Kelly, C. Clarke, M. Clynes, N. Barron","doi":"10.4155/PBP.14.28","DOIUrl":"https://doi.org/10.4155/PBP.14.28","url":null,"abstract":"Fundamental to the efficient production of quality biopharmaceuticals is the selection, optimization and tailored manipulation of the mammalian cellular production host. Engineering of these cell factories, predominantly the Chinese hamster ovary cell and advancements in bioprocess regimens have led to greatly increased product titres. The ability of miRNAs to regulate gene expression on a global level has generated considerable interest in these molecules as potential cell engineering targets. In this review, we briefly describe their organization and biogenesis and discuss their attributes as engineering tools in Chinese hamster ovary cells. The development of particular engineering strategies based upon further dissection of miRNA behavior will be considered, with particular emphasis on encouraging examples in Chinese hamster ovary cells and their potential for further development.","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"323-337"},"PeriodicalIF":0.0,"publicationDate":"2014-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.28","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sophisticated cell-free protein synthesis (CFPS) systems have been developed as an alternative to recombinant expression in cultured cells. In this review, we present advances in the field of mammalian-based CFPS by highlighting recently established systems derived from mouse fibroblasts, HeLa, hybridoma, CHO and K562 cells. We further highlight ongoing challenges in the field of mammalian-based CFPS, such as the optimization of already established platforms and the development of novel systems in order to further increase protein yields and reduce manufacturing costs while facilitating the synthesis of a huge number of biologically active target proteins. Advances in mammalian-based CFPS shall expand the number of future applications of CFPS in the area of pharmaceutical research and development.
{"title":"Developing cell-free protein synthesis systems: a focus on mammalian cells","authors":"A. K. Brödel, S. Kubick","doi":"10.4155/PBP.14.30","DOIUrl":"https://doi.org/10.4155/PBP.14.30","url":null,"abstract":"Sophisticated cell-free protein synthesis (CFPS) systems have been developed as an alternative to recombinant expression in cultured cells. In this review, we present advances in the field of mammalian-based CFPS by highlighting recently established systems derived from mouse fibroblasts, HeLa, hybridoma, CHO and K562 cells. We further highlight ongoing challenges in the field of mammalian-based CFPS, such as the optimization of already established platforms and the development of novel systems in order to further increase protein yields and reduce manufacturing costs while facilitating the synthesis of a huge number of biologically active target proteins. Advances in mammalian-based CFPS shall expand the number of future applications of CFPS in the area of pharmaceutical research and development.","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"101 1","pages":"339-348"},"PeriodicalIF":0.0,"publicationDate":"2014-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.30","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Valentin Jossen, S. Kaiser, Carmen Schirmaier, J. Herrmann, A. Tappe, D. Eibl, A. Siehoff, C. Bos, R. Eibl
Background: To improve cultivation conditions for human bone-marrow-derived mesenchymal stem cells, we redesigned the commercially available UniVessel® SU bioreactor using results obtained from computational fluid dynamics. The goal was to produce ≥1 × 109 cells and to achieve expansion factors ≥30. Screening studies suggested that microcarrier solid fractions of at least 0.3% are required to reach the appropriate cell densities. Results: The fluid flow pattern found in the most promising modification (#2) was altered by increasing the impeller blade angle and lowering the off-bottom clearance. As a result, the maximum required specific power input was reduced by a factor of 2.2–4.6, depending on the microcarrier concentration, and peak cell densities were 3.4-times higher than in the standard version. Conclusion: The peak cell number of nearly 1.1 × 109 cells (expansion factor = 35), which was achieved in our low-serum cultivations, indicates an improvement in the redesigned UniVessel® SU configuration f...
{"title":"Modification and qualification of a stirred single-use bioreactor for the improved expansion of human mesenchymal stem cells at benchtop scale","authors":"Valentin Jossen, S. Kaiser, Carmen Schirmaier, J. Herrmann, A. Tappe, D. Eibl, A. Siehoff, C. Bos, R. Eibl","doi":"10.4155/PBP.14.29","DOIUrl":"https://doi.org/10.4155/PBP.14.29","url":null,"abstract":"Background: To improve cultivation conditions for human bone-marrow-derived mesenchymal stem cells, we redesigned the commercially available UniVessel® SU bioreactor using results obtained from computational fluid dynamics. The goal was to produce ≥1 × 109 cells and to achieve expansion factors ≥30. Screening studies suggested that microcarrier solid fractions of at least 0.3% are required to reach the appropriate cell densities. Results: The fluid flow pattern found in the most promising modification (#2) was altered by increasing the impeller blade angle and lowering the off-bottom clearance. As a result, the maximum required specific power input was reduced by a factor of 2.2–4.6, depending on the microcarrier concentration, and peak cell densities were 3.4-times higher than in the standard version. Conclusion: The peak cell number of nearly 1.1 × 109 cells (expansion factor = 35), which was achieved in our low-serum cultivations, indicates an improvement in the redesigned UniVessel® SU configuration f...","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"311-322"},"PeriodicalIF":0.0,"publicationDate":"2014-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.29","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background Human viral vaccine manufacturing formed the basis of using animal cell technology for biopharmaceuticals in the 1960–1970s, replacing products derived from animals or human blood [1]. The majority of recombinant protein products, such as hormones and blood factors, made this transition from mammalian to a recombinant source, and later, being relatively well-characterized products, adopted stringent regulatory guidelines [2] based on scientific understanding. This also led to the use of a limited number of standard target expression systems to generate a product with specific predefined product characteristics and quality (CHO, Escherichia coli, Saccharomyces or Picchia). In contrast to recombinant biopharmaceutical proteins, the present situation for viral vaccines is characterized by a lack of standardization and diversity in expression systems. This diversity is further enhanced by the various approaches followed in viral vaccine development. Although recombinant subunit products to generate viral vaccines, such as virus-like particles (hepatitis B and human papillomavirus) and virosomes (Inflexal V; Crucell, The Netherlands), have reached the market, the majority of viral vaccines, as discussed in this paper, still takes the production of viruses (split, inactivated or live attenuated) as a starting point. Since most vaccines are given to healthy children, the introduction of new cell lines in viral vaccine production has been a low priority compared with product safety. Therefore, manufacturers may have selected cell lines based on conservative approaches, while tolerating potential inefficiencies. However, recent endeavors in modernization of classical (e.g., influenza and polio) and new (e.g., respiratory syncytial virus) viral vaccines have initiated exploration of exciting new viral expression systems [3].
{"title":"How to choose the correct cell line for producing your viral vaccine: what is important?","authors":"L. V. D. Pol, W. Bakker","doi":"10.4155/PBP.14.19","DOIUrl":"https://doi.org/10.4155/PBP.14.19","url":null,"abstract":"Background Human viral vaccine manufacturing formed the basis of using animal cell technology for biopharmaceuticals in the 1960–1970s, replacing products derived from animals or human blood [1]. The majority of recombinant protein products, such as hormones and blood factors, made this transition from mammalian to a recombinant source, and later, being relatively well-characterized products, adopted stringent regulatory guidelines [2] based on scientific understanding. This also led to the use of a limited number of standard target expression systems to generate a product with specific predefined product characteristics and quality (CHO, Escherichia coli, Saccharomyces or Picchia). In contrast to recombinant biopharmaceutical proteins, the present situation for viral vaccines is characterized by a lack of standardization and diversity in expression systems. This diversity is further enhanced by the various approaches followed in viral vaccine development. Although recombinant subunit products to generate viral vaccines, such as virus-like particles (hepatitis B and human papillomavirus) and virosomes (Inflexal V; Crucell, The Netherlands), have reached the market, the majority of viral vaccines, as discussed in this paper, still takes the production of viruses (split, inactivated or live attenuated) as a starting point. Since most vaccines are given to healthy children, the introduction of new cell lines in viral vaccine production has been a low priority compared with product safety. Therefore, manufacturers may have selected cell lines based on conservative approaches, while tolerating potential inefficiencies. However, recent endeavors in modernization of classical (e.g., influenza and polio) and new (e.g., respiratory syncytial virus) viral vaccines have initiated exploration of exciting new viral expression systems [3].","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"207-210"},"PeriodicalIF":0.0,"publicationDate":"2014-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.19","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347232","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The understanding of the nutritional requirements for Chinese hamster ovary (CHO) cells and other immortalized cell lines was an early milestone in developing cell culture media. A key aspect of these early studies was defining those amino acids termed ‘essen tial’ for survival and growth of different cell lines. However, the amino acids essential for growth of cells in culture differ from those defined as essential in biochemistry texts.
{"title":"Are nonessential amino acids not so redundant for Chinese hamster ovary cell lines","authors":"Dina Fomina-Yadlin, J. McGrew","doi":"10.4155/PBP.14.14","DOIUrl":"https://doi.org/10.4155/PBP.14.14","url":null,"abstract":"The understanding of the nutritional requirements for Chinese hamster ovary (CHO) cells and other immortalized cell lines was an early milestone in developing cell culture media. A key aspect of these early studies was defining those amino acids termed ‘essen tial’ for survival and growth of different cell lines. However, the amino acids essential for growth of cells in culture differ from those defined as essential in biochemistry texts.","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"211-213"},"PeriodicalIF":0.0,"publicationDate":"2014-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.14","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70346574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xiaofeng Dai, Wenwen Guo, Quan Long, Yankun Yang, L. Harvey, B. McNeil, Zhonghu Bai
Prediction of soluble protein expression levels in Escherichia coli based on the nature of protein itself remains a challenge for bioprocess development (BD). This review will critically discuss the current efforts and achievements that employ computational approaches to develop prediction models for soluble protein expression in E. coli. The contrast between the remarkable progresses made on the predictive models achieved by bioinformatics and their relatively infrequent application in BD will be explained. The effects of process-relevant variables at four different levels on the expression of heterologous proteins, for example, gene, vector, host cell and cultivation process, and also a critical comparison of several established bioinformatics tools for predicting expression levels will be presented. The potential utility of this emergent technology to increase the efficiency of BD strategies and thereby to reduce the cost of establishing a process for soluble protein expression are critically examined.
{"title":"Prediction of soluble heterologous protein expression levels in Escherichia coli from sequence-based features and its potential in biopharmaceutical process development","authors":"Xiaofeng Dai, Wenwen Guo, Quan Long, Yankun Yang, L. Harvey, B. McNeil, Zhonghu Bai","doi":"10.4155/PBP.14.23","DOIUrl":"https://doi.org/10.4155/PBP.14.23","url":null,"abstract":"Prediction of soluble protein expression levels in Escherichia coli based on the nature of protein itself remains a challenge for bioprocess development (BD). This review will critically discuss the current efforts and achievements that employ computational approaches to develop prediction models for soluble protein expression in E. coli. The contrast between the remarkable progresses made on the predictive models achieved by bioinformatics and their relatively infrequent application in BD will be explained. The effects of process-relevant variables at four different levels on the expression of heterologous proteins, for example, gene, vector, host cell and cultivation process, and also a critical comparison of several established bioinformatics tools for predicting expression levels will be presented. The potential utility of this emergent technology to increase the efficiency of BD strategies and thereby to reduce the cost of establishing a process for soluble protein expression are critically examined.","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"253-266"},"PeriodicalIF":0.0,"publicationDate":"2014-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.23","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Plant biotechnology may not be a familiar concept to the general public, but it is a rapidly developing field of research that involves the use of plants, plant tissues and plant cell cultures to make or modify products and processes. The versatility of plants and plant cells can be harnessed to produce diverse products, including valuable proteins. This is often described as ‘molecular farming’ and it requires the introduction of foreign DNA into plants or plant cells, turning them into factories for the production of specific recombinant protein products. The term ‘molecular pharming’ is often used instead to highlight the production of protein-based biopharmaceuticals, which contributes to the sustainable production of drugs that promote human and animal wellbeing. Both terms also apply to the production of valuable secondary metabolites such as the anticancer drugs paclitaxel, vincristine and vinblastine, but we will focus on recombinant proteins and their use as biopharmaceuticals in this article. The biopharmaceutical markets have expanded rapidly over the last 20 years, and are projected to more than double in volume over the next decade from US$200 billion in 2013 to at least US$500 billion in 2020. The two major biopharmaceutical production systems are microbes (mainly Escherichia coli and yeast) and mammalian cells such as the Chinese hamster ovary platform. In both cases, productivity has increased substantially over the last decade due to process optimization, platform standardization and genetic improvements. Both the US FDA and European Medicines Agency are familiar with these systems, and standard protocols can be followed to ensure the approval of new products. However, equivalent protocols are only just emerging for plant-based production systems, and only one plant-derived biopharmaceutical protein is currently on the market. With their established production infrastructure and regulatory framework, microbial and mammalian production systems have raced far ahead of their plant-based counterparts. No company will change their production host without a clear economic benefit, nor will they consider plants and plant cells for new products if there is no advantage over their incumbent technology. Furthermore, new companies will not base their manufacturing on a second-best option. Therefore, plant-based systems must begin to compete head-to-head with the established systems and, on a technological basis, we can already identify the areas where plantbased systems have the advantage, namely in terms of speed, improved product quality and scalability. The international success story of molecular pharming began in 2006 with the US Department of Agriculture approval of a poultry vaccine against Newcastle disease developed by Dow AgroSciences (IN, USA) [1,2]. The vaccine was manufactured in transgenic tobacco cell suspension cultures and was a benchmark for the regulatory acceptance of plants as a manufacturing platform, Molecular pharmin
{"title":"Molecular pharming in plants and plant cell cultures: a great future ahead?","authors":"A. Ritala, S. Häkkinen, S. Schillberg","doi":"10.4155/PBP.14.21","DOIUrl":"https://doi.org/10.4155/PBP.14.21","url":null,"abstract":"Plant biotechnology may not be a familiar concept to the general public, but it is a rapidly developing field of research that involves the use of plants, plant tissues and plant cell cultures to make or modify products and processes. The versatility of plants and plant cells can be harnessed to produce diverse products, including valuable proteins. This is often described as ‘molecular farming’ and it requires the introduction of foreign DNA into plants or plant cells, turning them into factories for the production of specific recombinant protein products. The term ‘molecular pharming’ is often used instead to highlight the production of protein-based biopharmaceuticals, which contributes to the sustainable production of drugs that promote human and animal wellbeing. Both terms also apply to the production of valuable secondary metabolites such as the anticancer drugs paclitaxel, vincristine and vinblastine, but we will focus on recombinant proteins and their use as biopharmaceuticals in this article. The biopharmaceutical markets have expanded rapidly over the last 20 years, and are projected to more than double in volume over the next decade from US$200 billion in 2013 to at least US$500 billion in 2020. The two major biopharmaceutical production systems are microbes (mainly Escherichia coli and yeast) and mammalian cells such as the Chinese hamster ovary platform. In both cases, productivity has increased substantially over the last decade due to process optimization, platform standardization and genetic improvements. Both the US FDA and European Medicines Agency are familiar with these systems, and standard protocols can be followed to ensure the approval of new products. However, equivalent protocols are only just emerging for plant-based production systems, and only one plant-derived biopharmaceutical protein is currently on the market. With their established production infrastructure and regulatory framework, microbial and mammalian production systems have raced far ahead of their plant-based counterparts. No company will change their production host without a clear economic benefit, nor will they consider plants and plant cells for new products if there is no advantage over their incumbent technology. Furthermore, new companies will not base their manufacturing on a second-best option. Therefore, plant-based systems must begin to compete head-to-head with the established systems and, on a technological basis, we can already identify the areas where plantbased systems have the advantage, namely in terms of speed, improved product quality and scalability. The international success story of molecular pharming began in 2006 with the US Department of Agriculture approval of a poultry vaccine against Newcastle disease developed by Dow AgroSciences (IN, USA) [1,2]. The vaccine was manufactured in transgenic tobacco cell suspension cultures and was a benchmark for the regulatory acceptance of plants as a manufacturing platform, Molecular pharmin","PeriodicalId":90285,"journal":{"name":"Pharmaceutical bioprocessing","volume":"2 1","pages":"223-226"},"PeriodicalIF":0.0,"publicationDate":"2014-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4155/PBP.14.21","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70347400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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