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High-Throughput Screening System

Until recently, the numerous cell lines of the ESETEC^® expression system were generated and cultivated by hand: Now several robotic systems handle cell-line development and screening on a micro- and milliliter scale.

High Speed Identification of Superior Production Strains

Wacker Biotech offers a series of proprietary bacterial E. coli strains that produce biologics for its customers. But which cell line is the best fit for the respective biologic? A screening system can now assist with this often difficult selection process.

“Combined with the necessary controls, we simultaneously screen about 1000 cell lines. This wouldn’t be possible without automation.”

Dr. Philipp Schmid, Senior Scientist Process Development, Wacker Biotech

Dr. Aurelia Stangl works in the biotechnology department of the Consortium, WACKER's corporate research unit. In the initial screening, she and her team test six ESETEC^® host strains, each of which is combined with 50 different expression plasmids.

Biopharmaceuticals, or biologics, are produced in living cells such as the common E. coli bacterium, a coliform bacterium that occurs naturally in the intestinal tract of many warm-blooded animals, including humans. But not all E. coli bacteria are alike. There are many different strains and, when combined with different expression plasmids needed for production, this leads to a wide range of potential cell lines. And not every cell line is suitable for every biologic. For that reason, one question has to be answered at the beginning of each production process: Which cell line is right for the biopharmaceutical?

Wacker Biotech, a subsidiary of the WACKER Group, is a contract manufacturing organization for biologics that uses, among other tools, a special expression and secretion system for producing protein-based biopharmaceuticals. Known as ESETEC^®, the patented system was developed on the basis of an E. coli K12 strain and a series of highly efficient expression plasmids. Thanks to ongoing optimization, ESETEC^® now has a formidable toolbox at its disposal, which consists of various ESETEC^® host strains and corresponding expression plasmids. “Identifying the best cell line for recombinant production of a pharmaceutical protein at the desired quality and quantity is a bit like looking for a needle in a haystack,” says Dr. Marcel Thön, who is responsible for cell-line development at Wacker Biotech.

Until now, the cell line development at Wacker Biotech was mainly driven by an experience-and knowledge-based approach. Depending on the target protein, certain basic ESETEC^® host strains and expression plasmids are excluded, while testing is carried out on others. The focus here relies on the initial screening of ten to twenty combinations of basic ESETEC^® host strains and expression plasmids, testing them on a milliliter scale in shake flasks.

300 cell lines are screened three times before the best suited combination for the production of a particular pharmaceutical protein is identified.

All the robotic systems used are customized one-offs, with some components made especially for the HTS.

In order to improve the selection process, the WACKER Consortium, i.e., WACKER’s central R&D, developed a multistage system designed to automatically select the right cell line: the High-Throughput Screening system, or HTS for short.

“We started developing our HTS in late 2019. Now it’s available for customer projects at Wacker Biotech,” says Dr. Philipp Schmid, who worked with an interdisciplinary team at the Consortium to establish the system. Expert groups from Corporate R&D in the fields of biotechnology, fermentation and analytics were involved in the project, as were employees from Advanced Analytics at WACKER’s Burghausen site. Wacker Biotech incorporated the perspectives of future customers into the project as well.

The system initially generates up to 300 different cell lines. In each case, six ESETEC^® host strains are combined with 50 different expression plasmids. “We also included an expression plasmid encoded with the customer’s target protein for each cell line produced from the various ESETEC^® host strains. Since every plasmid itself has a variety of genetic properties, you end up with a lot of different possible combinations,” Schmid explains.

K12 is the name of the E. coli bacterial strain on the basis of which the ESETEC^® expression system was developed.

Automated Analysis

New solutions also had to be found to achieve the throughput. Thus, the space required for cell line preparation was reduced to one-twelfth.

The first step is to determine the productivity of each of the 300 combinations. For a better comparison, three replicates are performed for each combination. “Combined with necessary controls, we simultaneously screen about 1,000 cell lines. You couldn’t do that without automation. It would take way too long,” Schmid goes on to say. Instead of generating and cultivating the strains by hand as done previously, cell line production and the initial screening are now performed automatically by multiple robotic systems on a sub-milliliter scale. Also, the productivity analyses are carried out with a newly established in-house method, i.e., automated RapidFire^® mass spectrometry – which needs just a few seconds per sample.

After the initial screening, the second step is to mimic the typical fermentation environment in miniature bioreactors for the eight to 16 most promising cell lines. This involves precisely controlling the pH, the temperature and the supply of oxygen and nutrients. The aim is to figure out which of the selected cell lines possess the best production properties under controlled conditions – a step that is likewise automated on a milliliter scale.

“We have provided the proof of concept. Now our customers can benefit from the system.”

Dr. Marcel Thön, Senior Expert, Bioprocess Development, Wacker Biotech

Last Step in the Laboratory

As soon as the robotic system is loaded with all consumables, manual intervention is only necessary at defined steps. This reduces the workload and creates capacity for innovation.

Once the system has identified the most productive bacteria, the team proceeds to the final step: classic fermentation in the laboratory. They also take closer look at the productivity of the remaining four to eight most promising candidates, studying them on a five-liter scale. In the end they have a winner that yields the best results in terms of quality and quantity.

The system has already demonstrated what it is capable of in actual practice. “We conducted multiple test series to show that automated identification of the most productive strain really works,” Schmid reports. In one example, the team was able to identify cell lines that did a better job of producing an antibody fragment that WACKER has used for years as a model protein for research purposes (see graphs).

The process allowed Wacker Biotech to revisit a protein that it had already been producing for a customer for several years, identifying new, more productive cell lines yielding an approximately 40 percent higher product titer.

“We delivered the proof of concept. Now our customers can benefit from the system. For them, the high throughput of the HTS greatly increases their chances of identifying the truly best combination of ESETEC^® host strain and expression plasmid for their target protein. Plus, we don’t lose as much time searching for the top performer,” says Thön.

Development of the high-throughput screening system is ongoing, including, for example, the integration of new ESETEC^® host strains and expression plasmids. Planning is also underway for using the system to screen for E. coli FOLDTEC^® cell lines. FOLDTEC^® is WACKER’s patented protein refolding technology.

Results from an HTS run using an antibody fragment as the target protein

All of the automatically generated cell lines were first cultivated on a sub-milliliter scale, and their productivity was analyzed using RapidFire^® mass spectrometry.

The top eight candidates were selected for cultivation under controlled conditions in a miniature bioreactor before final productivity studies were conducted on the best remaining candidates.

Both the controlled cultures in the mini-bioreactor as well as the final laboratory-scale fermentation studies showed that the selected ESETEC^® cell lines #2 and #4 were more productive than the control cell line, which had been optimized using a knowledge-based approach over several years.