Improved selection of protein-producing cell lines using a scale-down approach – a case study

The selection of a stable protein expressing cell line is a tedious process in which two substantially different approaches are used.
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Written by expert: Alfred, Global Technical Sales Director

The selection of stable protein producing cell lines is tedious, time consuming and capital intensive. The introduction of various high-throughput cell screening platforms and robot-assisted clone pickers is reducing reliance on harsh selection conditions and making it faster and easier to isolate the rare high-producing clones from thousands of transfected cells. However, this is only half the battle. Once candidate clones have been selected based on protein yields, there is still the hard work of assessing their suitability for commercial-scale production of the intended protein product. Extensive experimentation is needed to study the behavior of the cells under manufacturing like conditions, confirm biological activity of the protein product, and assess stability of the cells over at least 30, and preferably 60 cell passages in the absence of selection pressure. At conventional scales, the number of experiments can quickly become unmanageable, and it can be difficult to obtain enough research material. This challenge only becomes more daunting as evolving technologies allow isolation of larger numbers of candidate clones.

Scaled-down platform for high-throughput analysis

At Batavia, we have tackled these challenges by developing SCOUT®—a high-throughput platform that faithfully mimics full-scale cell culture and protein purification steps in miniature. This effectively down-scales the whole manufacturing process, so that the performance of many clones can be assessed, and their respective proteins purified under manufacturing like conditions—at small scale, at the same time, and in one system. By combining innovative scale-down technologies with intelligent experimental design and powerful multivariate data analysis, SCOUT allows the selection of top protein producer clones based on all crucial selection criteria: yield, biological activity, glycan structure, cell growth, population doubling time, and cell density. The SCOUT system makes use of small-scale bioreactor tubes (2-20 mL working volume). Subsequently, a high throughput purification technology is utilized, using purification plates, spin filters and spin traps. This production platform is fully integrated with Batavia’s analytical capabilities to quickly gain pivotal insight in protein production and cell behavior.

Case study: stable protein expressing cell line for EPO

The SCOUT technology was used in a program aimed to generate a high expressing recombinant erythropoietin (EPO) expressing CHO cell line. Starting with 250 clones, it took the team about 5 weeks to select 40 top-performing cell lines. The selection was based on excellent population doubling time (17-20 hours) and cell growth (up to 1×107 cells per mL). The protein product from each cell line was then purified in a single-step process with 60-70% recovery. This delivered approximately 500-800 μg EPO from each 20-mL cell culture. This material proved more than sufficient for subsequent product characterization studies, which included charge analyses by IEF, NP-HPLC, AEX-HPLC and MALDI-MS. In addition, purified material was used to determine biological activity using an in vitro assay with UT-7 cells.

In a timeframe of just 10 weeks, the team banked high EPO-producing cell clones (620 mg/L). All relevant glycan structures were present in the product, resulting in high bioactivity well inside the EPO marketed product release range. This example clearly shows the power of high-throughput, small scale manufacturing technology to aid cell selection and quickly anchor multivariate manufacturing processes reducing timelines and costs.

We are dedicated to help bring biopharmaceuticals to the market at higher speed, with reduced costs, and with a higher success rate. Batavia Biosciences has vast experience in developing stable protein expressing cell lines. Our experienced protein experts are well equipped to take on any challenge associated with production of protein-based pharmaceuticals.


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