New technologies will add flexibility, extend circulatory half-life
In recent years there has been an increasing demand for safer human therapeutics, leading to major developments in bioprocessing. Animal-free components offer an ethical, safe solution for the production of ingredients that form the basis of biological products. These ingredients are gaining popularity as regulatory authorities begin to implement strict quality measures on products to improve safety, particularly with potential contamination risks from pathogens such as viruses in animal-derived ingredients. The development and application of animal-free solutions for the production of biopharmaceuticals has many safety and regulatory advantages and in addition is economically viable and commercially scalable.
The introduction of yeast-based protein expression systems provides the advantages of stable, high-level protein expression using animal component free materials delivering a highly competitive cost of goods. Application of these expression systems to protein engineering technologies provides a powerful solution package for the industry.
Saccharomyces cerevisae is a species of budding yeast most commonly used in industries such as beer and bread. It has an extensive and successful track record in the production of protein therapeutics, such as insulin. When researchers investigate organisms to use in their studies, they look for several qualities including size, generation time, accessibility, manipulation, genetics, conservation of mechanisms and potential economic benefit.
Saccharomyces cerevisae has developed as a model organism because it scores positively on a number of these traits. Saccharomyces cerevisae is small with a quick generation time and can be easily cultivated, allowing for the swift production and maintenance of many specimen lines at low cost. It is also easily manipulated and can be transformed allowing for either the addition of new genes or deletion through homologous recombination. In addition, the ability to grow the yeast organism as a haploid simplifies the creation of gene knockout strains. As a eukaryote, Saccharomyces cerevisae also shares the complex internal cell structure of plants and animals without the high percentage of non-coding DNA that can confound research in higher eukaryotes.
A series of Saccharomyces cerevisae strains have been developed with various desirable traits including genetic stability, high copy number expression plasmids, protease deficient mutants and strains deficient in the enzymes involved in
Protein Expression Systems
Expression systems are systems for producing proteins as a result of the expression of recombinant genes encoding the desired polypeptide. These genes are introduced into expression hosts, using vector systems that enable the gene to be maintained and expressed, allowing analysis of protein structure and function. Use of recombinant proteins varies widely from functional studies in vivo to large-scale production for structural studies and therapeutics.
Yeast expression systems have proven to be extremely useful for the expression and analysis of eukaryotic proteins, providing a flexible, controllable and effective solution. Protein expression in yeast offers a number of advantages, such as high yield protein expression, high productivity, rapid high cell density growth and stable product strains, and is genetically well characterized. It is also highly cost-effective when compared to insect or mammalian cells.
The Saccharomyces cerevisiae-based expression system is optimized for the production of recombinant proteins where glycosylation does not naturally occur or can be designed without impacting product efficiency. Problems that are associated with prokaryotic alternatives — such as improper disulfide bond assignment, risks of protease degradation and inclusion body formation — can be overcome. Success in producing difficult to express proteins such as fully functional transferrin further shows the utility of these strains.
Albumin Fusion Technology
Albumin fusion technology is utilized for producing albumin joined to therapeutic protein or peptide, defined at the genetic level. Albumin fusion proteins are manufactured using proprietary yeast-based protein expression systems. This technology offers the ability to make completely new therapeutics that were previously out of reach. It is a simple, flexible platform for the production of proteins with extended circulatory half-life, resulting in less frequent administration and increased bioavailability.
Albumin is a natural, abundant carrier molecule, present in high volumes in the bloodstream and has a naturally long half-life of 20 days. It is an ideal, effective carrier for transporting various molecules around the body and has no endogenous activity. By fusing pharmaceutical proteins to albumin via albumin fusion technology, a protein’s half-life can increase from minutes to hours and hours to days.
Figure 1: Albumin fusion leading to reduced dosing frequency
and sustained exposure (schematic)
There are several binding points on the heart-shaped albumin molecule, specifically at either C- or N- termini, where peptides and proteins can be placed. It is even possible to fuse two different proteins together, with the use of albumin, in the same recombinant molecule to give two different functions at the same time. As a result, the new technology is not only suitable for improving existing product but also for making new products such as bi-functional proteins.
Albumin fusion technology offers a number of advantages such as increased half-life of the therapeutic protein. This confers advantages for patients such as reduced dose rates and reduced side effects, leading to improved stability and shelf life of the proteins (see Fig 1 above).
A considerably reduced dose means that the treatment is much more cost-effective, which is becoming significantly important with the increasing focus on healthcare costs and accessibility to medicine. A lower dose also leads to reduced side effects as the toxicity level of the protein may not be reached, meaning the drug dose remains within the therapeutic range, increasing the patients’ tolerance to the drug. Additionally, it has been demonstrated that albufuse-based molecules can lead to more favorable tissue distribution within the body reducing the risk of localized retention at the site of administration.
In addition, the yeast expression system provides a highly consistent and reliable supply of the therapeutic protein of interest, a major concern for companies as they develop their products through clinical trials and to market.
With increasingly stricter safety regulations being placed on biopharmaceutical companies, the demand for products that consist of animal-free ingredients is increasing. Yeast expression systems and proprietary protein engineering technologies offer the industry tools with which to develop products free of animal-derived ingredients. By implementing yeast expression systems, companies are able to develop a fully defined, animal-free bioprocess which in addition to offering many health and safety advantages is also more economically feasible and commercially scalable.
Svend Licht is senior director, Bioprocess Technologiesat Novozymes Biopharma. He can be reached at email@example.com.