Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

Abstract

From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

Raman microspectroscopy for the development and screening of recombinant cell lines

“…The authors demonstrated that Raman spectroscopy is capable to discriminate producing and nonproducing CHO cells based on their biochemical fingerprints, which were detected from living single cells without the use of external labels. Classical methods for the identification and selection of recombinant mammalian high-producer cell lines are laborious and time-consuming, and often focus on invasive upstream process analyses [6,7]. After cell transfection with the expression vector and cell growth under selection pressure, state-of-the-art methods require minimal dilution cloning procedures on the heterogeneous cell pool to allow the formation of single clonal cell colonies [7].…”

“…Classical methods for the identification and selection of recombinant mammalian high-producer cell lines are laborious and time-consuming, and often focus on invasive upstream process analyses [6,7]. After cell transfection with the expression vector and cell growth under selection pressure, state-of-the-art methods require minimal dilution cloning procedures on the heterogeneous cell pool to allow the formation of single clonal cell colonies [7]. Then, in most cases, clonal cell colonies are picked manually, which are then transferred into a new culture vessel and expanded for several weeks, before subsequent analysis of protein titers can be performed.…”

“…Then, in most cases, clonal cell colonies are picked manually, which are then transferred into a new culture vessel and expanded for several weeks, before subsequent analysis of protein titers can be performed. To guarantee cellular monoclonality, several rounds of single cell sub-cloning are performed successively [7]. Although picking of clonal cell colonies can be automatized, the probability to identify the ideal high-producer cell clone is limited by the number of clones that can be selected [8].…”

“…The introduction of fluorescent reporter genes is a possible method that can facilitate the use of high-throughput single cell screening techniques such as flow cytometry. However, the expression of an additional fluorescent protein can change cell metabolism, decrease cell proliferation rates and reduce product yields [7]. Expression levels of fluorescent reporter proteins can change over culture time and do not necessarily correlate to the production or secretion quantity of the protein product, thus, flow cytometry is not ideal for cells that secrete the protein product into the media [7].…”

“…However, the expression of an additional fluorescent protein can change cell metabolism, decrease cell proliferation rates and reduce product yields [7]. Expression levels of fluorescent reporter proteins can change over culture time and do not necessarily correlate to the production or secretion quantity of the protein product, thus, flow cytometry is not ideal for cells that secrete the protein product into the media [7]. In addition, fluorescent reporter proteins might also be secreted by the cells into the media and could impede the purification of the protein product in down-stream processing.…”