Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.     

构建生物药物分析实验教学改革新模式

生物药物分析实验是生物药物分析理论课程的重要补充,是培养学生掌握和巩固理论知识以及实验操作技能的重要途径。为适应现代高等教育对本科生的培养要求,对生物药物分析原有单一的实验教学模式进行改革已势在必行。我学院生物药物分析实验教学改革从实验教学内容、实验教学方法、实验教学手段等方面进行实践探索,建立了“验证性．综合设计性实验教学”、“科研一实验教学相结合”、“开放式实验教学”等新型的实验教学模式,在近几年的本科实验教学活动中,对于培养学生的实际操作能力、独立分析问题和解决问题的能力,团队协作等方面的能力起到了明显的效果,受到学生好评。     

Ultra-deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in Chinese hamster ovary cells

Recent sequencing of the Chinese hamster ovary (CHO) cell and Chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering.     

基于Delphi法的中国生物制药行业技术发展趋势预测分析

运用传统的技术预测方法---Delphi法,通过组织“我国生物医药技术发展趋势预测”专家调研,对我国生物制药行业的技术发展方向进行了预测分析,目的是从技术发展的角度给出生物制药行业投资机会分析的一些可供借鉴的建议。调研结果显示,我国生物药品的研发应采取跟踪模仿与创新相结合的模式,进行模仿性、延伸性的新药研发,重点开发肿瘤药物、疫苗和诊断试剂等,重点开发针对恶性肿瘤、心脑血管疾病及感染性疾病(炎症)等病症的药物;目前影响我国生物药品技术发展的主要因素包括宏观政策、市场需求、资源特性、新药审批制度等;在未来生物药品新药研发过程中最有可能被采用的新技术、新方法包括细胞凋亡机制、计算机辅助设计及人类基因组研究等。     

Improved cytosolic delivery of macromolecules through dimerization of attenuated lytic peptides

Intracellular delivery of biomacromolecules is a challenging research field in chemical biology and drug delivery. We previously reported a peptide named L17E, which successfully delivered functional proteins, including antibodies, into cells. However, relatively high concentrations of L17E and proteins are needed. In this study, we prepared dimers of L17E and its analog L17E/Q21E. Dimerization of L17E increased cytotoxicity leading to reduced intracellular delivery compared with L17E. On the other hand, the dimers of the L17E analog, L17E/Q21E, especially when tethered at the N-termini, yielded a comparable level of intracellular delivery with L17E at decreased amounts of delivery peptides and cargoes.     

Continuous bioprocessing: The real thing this time?

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides

Recent studies have shown that most peptide sequences encrypted in food proteins confer bioactive properties after release by enzymatic hydrolysis. Such bioactivities, which include antithrombotic, antihypertensive, immunomodulatory and antioxidant properties, are among the traits that are of biological significance in therapeutic products. Bioactive peptides could therefore serve as potential therapeutic agents. Moreover, research has shown that peptide therapeutics are toxicologically safe, and present less side effects when compared to small molecule drugs. However, the major conventional methods i.e. the synthetic and biotechnological methods used in the production of peptide therapeutics are relatively expensive. The lack of commercially-viable processes for large-scale production of peptide therapeutics has therefore been a major hindrance to the application of peptides as therapeutic aids. This paper therefore discusses the plausibility of manufacturing pharmaceutical-grade bioactive peptides from food proteins; the challenges and some implementable strategies for overcoming those challenges.     

Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system

Demand is increasing for therapeutic biopharmaceuticals such as monoclonal antibodies. Achieving maximum production of these recombinant proteins under developmental time constraints has been a recent focus of study. The majority of these drugs are currently produced in altered Chinese hamster ovary (CHO) cells due to the high viability and the high densities achieved by these cells in suspension cultures. However, shortening the process of developing and isolating high-producing cell lines remains a challenge. This article focuses on current expression systems used to produce biopharmaceuticals in CHO cells and current methods being investigated to produce biopharmaceuticals more efficiently. The methods discussed include modified gene amplification methods, modifying vectors to improve expression of the therapeutic gene and improving the method of selecting for high-producing cells. Recent developments that use gene targeting as a method for increasing production are discussed.     

Immunogenicity of therapeutic proteins. Part 2: impact of container closures

Immunogenicity as a potential consequence of therapeutic protein administration is increasingly being scrutinized in the biopharmaceuticals industry, particularly with the imminent introduction of biosimilar products. Immunogenicity is an important safety aspect requiring rigorous investigation to fully appreciate its impact. Factors involved in product handling, such as storage temperature, light exposure, and shaking, have been implicated in immunogenicity, while container closure systems are no less important. Intended to provide a stable environment for the dosage form, container closures may also interact with a product, affecting performance and potentially enhancing immunogenicity. Glass surfaces, air-liquid interfaces, and lubricants can mediate protein denaturation, while phthalates in plastics and latex rubber are sources of extractables and leachates that may contaminate a product, causing allergic reactions and increasing immunogenicity. The manufacture of therapeutic proteins therefore requires rigorous safety evaluations not just in the context of the product, but also product containment.