Biopharmaceutical benchmarks 2014

119

Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read based assemblies. Here, we completely resequenced C. griseus using Single Molecule Real Time (SMRT) sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important CHO cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.

Section: Introductionmentioning

confidence: 99%

A reference genome of the Chinese hamster based on a hybrid assembly strategy

Rupp

MacDonald

et al. 2018

119

“…They include blockbuster drugs such as Humira®, Avastin®, and Herceptin®, with the top five selling drugs generating nearly 70 billion USD in revenue in 2013 alone. Currently, biopharmaceuticals are predominantly produced using mammalian cell cultures to ensure appropriate glycosylation, with Chinese Hamster Ovary (CHO) cells the most prevalent host for the production of glycoproteins in industry today (Kyriakopoulos and Kontoravdi, 2013; Walsh, 2014). …”

Section: Introductionmentioning

confidence: 99%

Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Goers

Ainsworth

Goey

et al. 2017

Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

“…Chinese hamster ovary (CHO) cells are the most frequently used mammalian host for the production of biopharmaceuticals (Walsh, 2014). For a better understanding of the biological background and to improve the production system “omics”‐based research has given insights into cellular changes triggered by overexpression, higher growth rates or extracellular stimulus like hypothermic cultivation, butyrate treatment, or hyperosmotic conditions (Datta et al, 2013; Kildegaard et al, 2013; Kim et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Sommeregger

Mayrhofer

Steinfellner

et al. 2016

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.