Biopharmaceuticals are produced in living cells such as E. coli. But not all E. coli are alike. There are many different strains, and when combined with various expression plasmids needed for production, a wide range of potential E. coli strain/plasmid combinations are created.
Moreover, not every E. coli strain/plasmid combination is suitable for every biologic. For that reason, one question has to be answered at the beginning of each production process: Which combination is right for the biopharmaceutical target protein?
Wacker Biotech, a CDMO and subsidiary of the Wacker Group, 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 series of highly efficient expression plasmids.
As a result of ongoing optimization, ESETEC now has a toolbox at its disposal which consists of various basic host strains and corresponding expression plasmids. Identifying the best strain/plasmid combination for recombinant production of a pharmaceutical protein at the desired quality and quantity is a bit like looking for a needle in a haystack.
Until now, 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 an initial screening of 10 to 20 combinations of basic host strains and expression plasmids, testing them on a milliliter scale in shake flasks.
To improve the selection process, the Wacker Consortium, that is, Wacker’s central R&D, developed a multistage high-throughput screening (HTS) system designed to automatically select the right combination of host strain and expression plasmid. The system initially generates up to 300 different combinations. In each case, six host strains are combined with 50 different expression plasmids. Each expression plasmid carries the customer’s gene of interest that encodes for the target protein. Since every plasmid itself has a variety of genetic elements, many different combinations are possible.
The first step is to determine the productivity of each of the 300 different combinations. For a better comparison, three replicates are performed for each combination. Combined with necessary controls, the system simultaneously screens about 1,000 bacterial clones. This wouldn’t be possible without automation. It would take far too long.
Instead of generating and cultivating the different bacterial clones by hand, as was done previously, transformation of different strains with different expression plasmids as well as the initial screening are now performed automatically by multiple robotic systems working on a submilliliter scale. Also, the productivity analyses are carried out with a newly established in-house method, namely, 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 8 to 16 most promising bacterial clones.
This includes the precise controlling of the pH and the temperature, as well as the supply of oxygen and nutrients. The aim is to figure out which of the selected bacterial clones possess the best production properties under controlled conditions—a step that is also automated at milliliter scale.
Last laboratory step
Once the system has identified the most productive strain/plasmid combination, the team proceeds to the final step: classic fermentation at laboratory scale. They take a 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 identify one candidate that yields the best results in terms of quality and quantity.
The system has already demonstrated what it is capable of in actual practice. Multiple test series were conducted to show that automated identification of the most productive strain/plasmid combination works. In one example, the team was able to identify new combinations that did a better job of producing an antibody fragment that Wacker Biotech has used for years as a model protein for research purposes (Figures 1–3).
The process also provided an opportunity to revisit a protein that Wacker Biotech has been producing for a customer for some time: identifying new, more productive combinations yielding an approximately 40% higher product titer. The proof-of-concept was shown. The benefits: high throughput of the HTS system greatly increases the chances of identifying the best combination of host strain and expression plasmids for different target proteins. Plus, not as much time is lost searching for high performers.
Development of the HTS system is ongoing, including, for example, the integration of new host strains and novel expression plasmids. Planning is also underway for using the system to screen for E. coli FOLDTEC® strains and plasmids, which are key elements of Wacker Biotech’s patented protein refolding technology.
Philipp Schmid, PhD, is a senior scientist, process development, and Marcel Thön, PhD, is a senior expert, bioprocess development, at Wacker Biotech.