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Microbial Secretion Systems Boost Active Pharmaceutical Ingredient Yields

05.08.2008 | Editor: Anke Geipel-Kern

Research lab at the Wacker Biotech Research Center. By controlling factors such as temperature and oxygen supply, researchers can create optimal conditions for bacteria growth. (Pictures: Wacker Chemie)
Research lab at the Wacker Biotech Research Center. By controlling factors such as temperature and oxygen supply, researchers can create optimal conditions for bacteria growth. (Pictures: Wacker Chemie)

Wacker Chemie has developed a patented E. coli strain for extracellular production of pharmaceutical proteins. The system transports proteins in functional conformation through the outer cell membrane into the culture medium. This proprietary technology provides the basis for cost-efficient industrial-scale production of ultra-pure active biopharmaceutical ingredients.

Protein pharmaceuticals, which are produced using a biological process (biologics), have enormous growth potential. Cancer medication, growth factors and active ingredients used to treat multiple sclerosis can be produced in bacterial cells. According to EuropaBio’s 2007 Annual Report, biotech drugs account for around 20 percent of all medication currently on the market and 50 percent of medication which is now in the pipeline. Market studies indicate that the number of biopharmaceuticals on the market has more than doubled over the past ten years, and these products have a 23 percent growth rate in the EU.

Biotechnology shows its real strengths in the production of pharmaceutical products. In contrast to protein pharmaceuticals which are extracted from blood or tissue, the contamination risks associated with state-of-the-art biologics is virtually zero. The fact that the pharmacological effectiveness of the proteins can be enhanced by targeted modification of amino acid sequences is an added benefit.

Bacteria, yeast cells and mammalian cell cultures are all potential producers of pharmaceutical proteins. Escherichia coli bacteria are a popular choice. These bacteria have been well researched, they are easy to handle, and the genetic properties are well known. However, the main attraction of E. coli is rapid growth, limited nutrient requirements and the ability to produce proteins quickly in high titers (compared to mammalian cells). Good time/space yields are also achievable. The challenge is to turn the bacteria into reliable, cost-effective producers of the largest possible volumes of active ingredients. Overexpression of recombinant products often results in proteins that are not properly folded and that are stored inside the cells as inclusion bodies. A series of elaborate and cost-intensive refolding and purification steps are needed to recover the proteins.

Extracellular production

To facilitate the production of proteins and antibody fragments, Wacker Chemie has developed secretion technology for extracellular production. During fermentation, the desired, properly-folded protein product can be secreted directly into the fermentation medium. This technology greatly simplifies the purification process. The cells do not have to be broken open to filter out and activate the proteins, because the correctly folded proteins are released into the fermentation medium in active form. Purification begins with a less inhomogeneous material, and it takes far fewer steps to produce a pure product.

Recombinant protein yields are also higher with extracellular production, because product accumulation is not limited by the restricted volume in the cell interior (cytoplasm) or the periplasm. This significantly reduces downstream costs. The secretion technology consists of special expression plasmids and a patented E.coli K 12 strain which was developed in house. This strain is an E.coli K 12 derivative (Biosafety Level 1), and its genetic properties are completely understood. In May 2007, a genome sequencing project conducted by Wacker Chemie and Eurofins Medienomix/MWG Biotech to analyze Wacker’s in-house bacterial protein production strain was brought to a successful conclusion. Based on the results, it has been possible to accurately document the properties of the strain and to continually improve the production strain.

Two-stage process

In principle, expression takes place in a two-stage process.

Initially, Sec translocase is used to translocate the target product through the cytoplasmic membrane into the periplasmatic space. The nascent protein contains short signal sequences which facilitate translocation out of the cytoplasm. During this step, the signal peptide is split off to release the native product.

The next step is to secrete the correctly folded product from the periplasm through the outer membrane directly into the culture medium, and this is where the unique properties of the proprietary Wacker strain play a major role. Specific modifications produce efficient secretion of the recombinant protein.

The secretion technology has produced robust, reproducible results in large-scale fermentation (up to 4.5 cubic meters). Using simple cell separation, the target product can be isolated in a soluble, native and active form from the fermentation broth.

Wacker has developed a toolbox containing different expression plasmids with varying properties (e.g. different signal sequences). Several helper plasmids are used to optimize expression, solubility and secretion of the recombinant target product. These elements are introduced as a second gene on the helper plasmid, or they are encoded on the helper plasmid and added to the expression plasmid. Helper plasmids include cytoplasmic chaperones, components of the secretion apparatus, periplasmic chaperones and disulfide bridge formation factors.

Antibody fragment production

Biotech production of peptides is very challenging. It is however an attractive option, because chemical synthesis of large peptides involves a number of steps which cost a lot of money. Peptide proteolysis causes problems during biotech production of E. coli. To address the problem, Wacker has developed derivatives of the secretion strain in which the genes that code the proteases have been switched off, allowing the intact recombinant peptide to pass through the cell membrane into the culture medium. Wacker secretion technology has already produced a number of prokaryotic and eukaryotic proteins (up to 10 g/L). Successful production of functional human antibody fragments with correct disulfide bridges and yields in excess of two grams per liter has been a particularly noteworthy achievement.

Driving down costs

Manufacturing costs are a major consideration in biologics production systems. Wacker’s secretion systems are suitable for production of a whole range of proteins, enzymes, antibody fragments and peptides. Extracellular production simplifies purification and eliminates the need for elaborate refolding steps, making production more efficient and cost effective.

 

About Wacker Biotech

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