A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

Abstract

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a highthroughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1 , followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis [2][3][4] . Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 -10,000 clones can be screened per operation cycle with the current system setup.This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

High‐throughput screening of antibody‐expressing CHO clones using an automated shaken deep‐well system

“…[17][18][19] However, many of these screening platforms are based on a version of batch secretion in static culture and are subject to the same lack of correlation described by Porter et al, 10 so are best used for eliminating nonexpressing cell lines before starting a more predictive suspension fed-batch screen. Newer, more highly automated cell culture screening systems have been developed, [20][21][22][23][24][25][26][27] including the recently reported implementation of nanofluidics. 28 These methods can enable high throughput fed-batch screening for application in cell line development as well as for process development and media evaluation.…”

High‐throughput screening and selection of mammalian cells for enhanced protein production

“…In addition to flow cytometry and cell sorting, there are a number of other exciting ways in which researchers have [89] utilized fluorescence as a tool to isolate HP clones. One of the most promising areas involves the use of automated protocols that greatly reduce time and labor, while considerably increasing the throughput, efficiency, and accuracy of the screening process.…”

An 'omics approach towards CHO cell engineering

“…Techniques employed for clonal selection have been traditional limiting dilution cloning (Puck and Marcus, 1955), flow cytometry and cell sorting (Borth et al, 2000; Klapperstuck et al, 2009; Kumar and Borth, 2012; Lee and Lee, 2012; Nicolette et al, 2011; Pichler et al, 2011; Vanderbyl et al, 2001), and gel microdrop technology (Hammill et al, 2000). More recently, these techniques have been combined with automation to improve throughput and reduce the labor involved (Kacmar and Srienc, 2005; Shi et al, 2011). Recent advances in clonal selection based on quality of the bioengineered protein include high‐throughput analysis based on sialic acid content in the bioengineered glycoprotein (Markely et al, 2010; Park et al, 2010).…”