Microbial expression systems are widely used to manufacture diagnostic and therapeutic proteins, with insulin being the best-known example. However, the commercialization success rate for new drug developments is less than 3%, partly due to high development and manufacturing costs, long turnaround times, and challenging product safety profiles. Contract development and manufacturing organizations (CDMOs) are working on microbial expression system updates to prepare for future applications, including antibody fragments and therapeutic peptides.
While microbial protein production systems present cost-efficient alternatives to insect or mammalian cells, the commonly used E. coli strains often deliver only modest yields, accompanied by high levels of potentially immunogenic substances and process contaminations. Moreover, identifying robust, high-yield expression and purification conditions that meet patient safety requirements takes valuable time.
These challenges contribute to drug development risk and limit the current scope of therapeutic proteins produced in microbial systems. Now, CDMOs are jumping in to play a key role in updating microbial expression systems, addressing limitations in existing applications, and getting ready for the manufacturing of state-of-the-art therapies.
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A new approach toward a microbial expression factory
An optimal microbial expression factory for therapeutic proteins needs to master the magic triangle of lower costs, shorter timeframes, and increased safety. In addition, product-critical quality attributes must be achieved, including proper protein folding, correct subunit assembly, and low aggregation levels.
The team at KBI Biopharma, a globally operating CDMO, set out to design the perfect microbial production factory. Leveraging their combined expertise in genetic optimization, the scientists layered together more than 1,000 gene edits to create PUREcoli™, a true microbial expression system update.
“For each customer project, we can choose from a set of proprietary PUREcoli substrains, PUREplasmids™, and PUREmedia™ to develop an optimal microbial production workflow – from micro fermentation screening to scale-ups in tens or hundreds of liter bioreactors.”
Case study: How does the PUREcoli system address the limitations of microbial expression?
Recently, a comprehensive case study was conducted to collect data that proved increased productivity, lower costs, faster development time, and enhanced patient safety.
Increased productivity
Productivity is the product yield per cell. At high peak cell densities, PUREcoli strains were shown to express three times more protein per cell than traditional E. coli strains. This provides higher product quantities and a threefold better ratio of product to process impurities (e.g., endotoxin, host cell DNA, and protein) in the raw material. PUREcoli strains were also shown to grow to two- to threefold higher cell densities. Combining the attributes of three times the number of cells and three times the expression level within each cell generally increases protein yields by nearly a factor of ten.
In combination with the optimal choice of E.coli substrains and expression plasmids from the KBI Biopharma library, yields have been increased by as much as a factor of 40 for a protein expressed in inclusion bodies.
Higher productivity obviously reduces the cost per mg of product. But there is also an indirect effect on overall manufacturing costs and timeframes.
“If we start with more protein output, we’ll have enough material at our disposal for a more flexible, shortened purification scheme, including precipitation protocols that provide high purity, albeit at lower recovery rates,” Nordwald explained.
Faster development time
In each of the three case studies aimed at reducing the number of unit operation steps, PUREcoli-derived starting material enabled four steps to be saved, including chromatography steps. For example, a cytokine could be expressed in soluble form to such high yields that an acid precipitation step followed by a single column was sufficient to obtain protein at drug substance specification-level purity while significantly reducing process time.
“Another way to increase purity is to use a dedicated PUREcoli strain that promotes the secretion of the target protein into the periplasmic space, a compartment containing few potential contaminants. Releasing proteins from the periplasm while keeping the bacterial cell intact is a straightforward procedure that shortens purification protocols, as demonstrated in a customer project,” Nordwald added.
In addition, the periplasmic space contains enzymes that catalyze the correct folding and formation of disulfide bonds. Therapeutic proteins with disulfide bonds, especially antibody fragments, can be processed and folded correctly when the proteins are secreted into the periplasm. There is also potential for smaller amino acid molecules, namely peptides, to be secreted to the periplasmic space and diffused to the extracellular space, simplifying bioprocessing.
Removing genes to increase purity and safety
During the stringent genetic modifications performed to create the PUREcoli strains, specific genes were deleted, attenuated, or upregulated to improve product purity. For example, enzymes involved in incorrect post-translational modifications were removed, while enzymes contributing to product-critical quality attributes were upregulated.
In the case studies, the resulting proteins were extensively characterized by liquid chromatography-mass spectrometry (LC-MS), demonstrating that these product-related impurities were at low to undetectable levels.
A general reduction in unnecessary host cell proteins not required for protein expression improves the overall cell metabolism. This permits cell growth at lower oxygen levels, which is often a limiting factor in fermentation processes. By the same mechanism, the cells generate less heat, reducing the cooling requirements of the fermenters.
Foaming is also significantly reduced in PUREcoli strains—a major problem in microbial cell culture, as foam can clog gas filters and limit cell density. This allows the use of less antifoam agents, which must be removed in the subsequent purification process.
PUREplasmids are maintained in the expression strains without the need for antibiotics, further reducing process impurities. In addition, a number of virulence-related or immunogenic proteins, including adhesins and pilins, were deleted. Overall, the levels of host cell protein, host cell DNA, endotoxins, and other contaminants could be reduced by a factor of two to three, contributing to lower risk in clinical trials and, eventually, improving the chances for commercialization.
Increasing plasma half-life: replacing PEG with PAS
A frequent issue with recombinant therapeutic proteins is their limited half-life in plasma. Polyethylene glycol (PEG) is often added to increase the hydrodynamic radius of the protein and slow down clearance by kidney filtration. However, PEGylation—the process of adding PEG—is a complex process that requires at least three additional unit operation steps for coupling reaction, buffer exchange, and clean-up.
PUREcoli™ strains allow an efficient use of the PASylation technology. Here, multiple repeats of the three amino acids proline (P), alanine (A), and serine (S) are added to the expression plasmids, giving rise to PASylated proteins in the bacterial lysate.
PAS repeats form a disordered structure, providing a biological alternative to PEG in prolonging plasma half-life. PASylating eliminates the need for PEGylation and also attenuates the product heterogeneity often observed from PEG reactions.
In the study, PAS repeats added to either the N- or C-terminal end did not affect protein structure, as calculated by the AlphaFold prediction software, or protein activity, as demonstrated in suitable activity and binding assays. An N- and C-terminal double-PASylated biosimilar could be produced with eightfold higher yields than standard microbial strains.
“While standard E.coli strains can also add PAS sequences, the stretches are typically shorter, limiting the effect on protein stability. In addition, the PASylation process reduces protein yield, which is a problem when productivity is already low,” said Nordwald.
Beyond insulin: Expanding the total portfolio of microbial expression systems
The rationale for producing therapeutic and diagnostic proteins in microbial cells is mainly to lower cost of goods compared to eukaryotic systems. As demonstrated in the case study, PUREcoli can be used in existing manufacturing projects to enable higher productivity, simplify purification schemes, and improve safety. The option to use PASylation instead of PEGylation further streamlines manufacturing processes. Although complex therapeutic molecules such as bispecific antibodies will continue to be manufactured in mammalian cell lines, the features of the PUREcoli system, including secretion and disulfide bond formation, open the door to expanding the portfolio of proteins produced in microbial systems to advanced therapeutics such as antibody fragments and peptides.
Watch KBI’s recent webinar and learn how PUREcoli™ can improve your microbial therapeutic protein production processes.
Images Courtesy: KBI Biopharma