The Evolution of Protein Expression and Cell Culture - - BioPharm International


The Evolution of Protein Expression and Cell Culture

BioPharm International
Volume 20, Issue 10

With the advent of the transcriptomics and proteomics era, researchers are attempting to understand the fundamental mechanisms underlying the differences between the cell lines producing low and high amounts of the protein of interest. Seth et al. used the DNA microarrays and two-dimensional gel electrophoresis (2DE) for eleven GS-NS0 cell lines to show that major functional class changes between low and high producers are in protein synthesis and cell death/growth.28 A recent report showed that there is a positive correlation between the specific productivity and the ratio of heavy- to light-chain mRNA expression of an antibody produced by a GS-NS0 cell line. The report also showed that the use of Northern and Southern analyses can highlight the presence of any abnormal mRNA species (in terms of molecular size) and can also indicate if the functional integrity of the site of insertion of the transgene has been compromised.29 The set of information generated from the above mentioned techniques can serve as useful markers to identify the overproducers among various clones early in the clone selection phase and there is no doubt that such techniques will be used to a greater extent in the coming years. In addition to understanding the mechanisms of cell productivity, it is equally important to understand how the stability of gene expression is conferred to a clone. This area of protein expression is also not well understood and there is a clear need to determine the molecular markers that can help identify stable cell lines early in the project cycle. There is also a need to enhance the understanding, at the chromatin level, of the specific regions of the genome that confer genetic stability to a transgene as well as the transgene integration approaches that can specifically target these regions.


After a clone is selected, the next task is to develop the growth medium. Simple, defined media for bacterial systems have been developed since the initial days of biotechnology manufacturing. Such media contain a carbon source (e.g., glucose, glycerol), a nitrogen source, a phosphorous source, mineral salts, and buffering components. Complex media, using yeast extract, casein hydrolysate, etc., are also common. Compared to bacterial media, the cell culture medium is rather complex. In the past, fetal bovine serum (FBS) was an essential component for the propagation of mammalian cells. Many industrial mammalian cell growth media developed in the 1990s did not use serum. They did, however, include multiple serum fractions containing hormones, growth factors, lymphokines, cytokines, transport proteins, attachment proteins, serum albumin, and lipid supplements. Such a medium that is not supplemented with serum but includes the discrete protein and bulk protein fractions is commonly known as serum free medium. Due to bovine spongiform encephalopathy (BSE) concerns, there is a strong motivation to develop processes without any serum derivative. Such processes, often called animal-protein-free processes are currently the most prevalent and often form the bases of current platforms being developed by many companies. The media for these processes may contain complex ingredients like hydrolysates. The latest industrial development has been to create formulations that do not depend upon the inclusion of any complex raw materials. Such completely defined media have been successfully developed.30 It remains to be seen, however, if the industry will universally adopt the use of completely defined media, because the complex media ingredients are often believed to be the key determinants of productivity.

The cell culture medium typically contains glucose, amino acids, vitamins, bulk ions, lipids, phospholipid precursors, nucleotides, buffering components, protective agents like Pluronic surfactants, antioxidants, and reducing agents. For serum-free formulations, complex ingredients may include various serum supplements and protein molecules like insulin and transferrin. For animal-protein-free and completely defined media, water insoluble components like cholesterol are delivered to cells by carrier molecules like cyclodextrin.31 Scientists now have been able to successfully grow NS0 cells, which were originally thought to be cholesterol auxotrophs, without cholesterol.32 It also has been shown that the cholesterol dependence of cholesterol dependent NS0 cells is due to epigenetic silencing because of the methylation of the CpG-rich region upstream of the transcription start site of the gene Hsd17b7 in NS0 cells.33

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