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The need for improved half-life extension technologies
January 2013
by Mark Perkins, Novozymes  |  Email the author

Today's pharmaceutical market is facing a number of pressures, in particular relating to moving drugs through clinical trials and to market faster and more cost-effectively. This stems from ever-shrinking budgets and growing financial burdens throughout the industry. In recent years, the industry has witnessed a significant increase in the investment into the development of targeted, biological drugs, with companies looking to take these tailored, novel therapies to market faster than ever before. The increased interest into these therapies is as a result of their decreased side effects for patients, which makes them ideal for treating conditions where medication needs to be administered frequently, such as chronic diseases.
However, biological drugs are often hampered by their characteristically short half-lives, which means that once administered, they can be cleared from the body in a matter of minutes. As a result of this short half-life, patients with chronic conditions such as diabetes, hemophilia and neutropenia are often required to administer higher dosages more regularly, leading to likelihood of reduced compliance, higher costs and greater risks of side effects. Drugs with a promising therapeutic value are often limited by this factor. For this reason, the pharmaceutical and biotech sectors are paying increasing attention to half-life extension strategies, with a number of research institutes and academic papers noting the growing trend in developing technologies that extend and improve the circulatory half-life of peptides and proteins.
Responding to this issue, many researchers have concentrated their efforts in recent years into developing half-life extension technologies that modulate the serum half-life of protein-based therapeutics to desired specifications. However, while researchers have managed to successfully extend the actual serum half-life, they have not been able to design flexible protein half-lives to deliver the required pharmacokinetics.
Current strategies used for extending half-life are those that increase hydrodynamic volume (PEGylation) or those that use FcRn-mediated recycling (albumin fusions). Although real progress has been made in the creation of novel technologies that modulate serum half-life, the market is still actively searching for a solution that will allow companies to tailor their therapies in line with specific medical indications.
Recent advances in albumin fusion technology
Recent enabling half-life extension technologies are based on serum albumin, a natural, non-immunogenic plasma carrier protein. Albumin is an optimal material upon which to base half-life extension technologies due to its naturally long half-life of 19 days in humans, in comparison to protein therapeutics that are often cleared from the body in as little as hours. Apart from its size, it is the pH-dependent recycling through the neonatal FcRn receptor that protects albumin from renal clearance and is responsible for its extended half-life. Like IgGs, albumin is taken up by cells through nonspecific pinocytosis and is protected from intracellular degradation through pH-dependent binding to the FcRn receptor in acidic endosomes. This interaction allows albumin to then be recycled back to the cell surface where it is released into circulation due to the physiological pH of the blood.
It is the pH-dependent interaction between albumin fusion and the FcRn receptor that provides the basis for the latest advancements in albumin fusion technology. The understanding of its impact on albumin fusion half-life has enabled the engineering of this interaction with the potential to modulate albumin's half-life. Previous studies that altered this interaction have been shown to impact the pharmacokinetics of the IgG.
Applying these same kinetic principles, enabling half-life extension technologies are able to modulate protein half-life through construction of albumin variants with altered binding affinity to FcRn. With the ability to modulate albumin half-life, researchers are provided with the opportunity to tailor therapeutics to specific disease states and fine-tune their drug design, holding significant benefits for drug developers and patients alike.
Enabling technologies also produce more stable blood levels in patients, and a reduced risk of side effects at the associated lower dose rate means that the toxicity level of the protein may not be reached. Instead, these technologies allow the drug dose to remain within the therapeutic range, increasing the patient's tolerance to the drug.
Broad platform applicability
The current approach when using albumin as a half-life extension technology is to conjugate, or genetically fuse. Both of these methods can be equally as effective, depending on specific drug delivery requirements. Lysine, tyrosine and the free thiol residues of the albumin molecule are used for chemical conjugation to the drug product, with the free thiol at position 34 of albumin the most widely used conjugation route. This approach is particularly useful for peptides containing maleimide groups that specifically react with the free thiol, allowing for the formation of a stable thioether bond between albumin and the peptide.
Alternatively, proteins can be genetically fused to the N- or C-terminus or even to both ends of the albumin variant. Using a contiguous cDNA of the target protein or peptide with DNA encoding the albumin variant of choice allows the generation of protein fusions exhibiting the required binding characteristics. A yeast expression system provides a high-quality, consistent and reliable supply of the protein of interest when a genetic fusion is applied. 
In recent studies, a range of albumin protein fusions has been generated to test that the albumin variants maintain their modified FcRn binding affinity when fused to a protein or peptide. The variants chosen displayed a range of binding affinities from low affinity albumins (HSA K500A) to albumins with a 15-fold increase in receptor binding (HSA K573P). Antibody fragments fused at the C-terminus, N-terminus or bivalent forms, as well as fusions to small or large peptides were compared to unfused albumin variants for FcRn affinity by SPR using Biocore technology. All albumin fusions tested presented different receptor affinities, correlating to their unfused variants. Each showed the same differences in ScRn binding as the control rHSA variant.
As a result of the technology, proteins and peptides can be bound at either the C- or N-terminus or both. This creates fusion molecules with monovalent, bivalent or bispecific affinity. In addition to protein- or peptide-based drugs, the technology also serves as a delivery vehicle for small molecules, providing a broad scope of usability. It also enables construction of albumin variants with altered binding affinity to FcRn, making it possible to modulate half-life extension of a fused target protein, while offering drug developers enhanced flexibility and control.
Minutes to hours, hours to days
While the market demand for technologies that allow the development of drugs with novel properties continues to grow, researchers must develop solutions that provide manufacturers with competitive solutions. Due to the limited number of biological drugs available on the market, researchers are now looking to adapt and improve upon those that are available to them. In recent years, significant work has been dedicated to studying the half-life of drugs, and while researchers have been successful in lengthening the half-life of proteins and peptides, they have yet to find a way to tailor the pharmacokinetics of certain drugs to specific medical needs. Enabling half-life extension technologies provide a solution to these issues by proving a platform in which drug developers are able to fine-tune the half-lives of drugs to certain therapeutic conditions.
Commercially, the latest advancements in half-life extension technologies offer drug manufacturers the opportunity to establish a niche position in the market with flexible products that offer improved performance throughout the drug lifecycle. The ability to modulate and tailor the half-lives of drugs offers the potential to significantly improve quality of life for patients with chronic conditions through lower and less frequent dosage levels. This can lead to increased patient compliance and the possibility for patients to administer their own drugs. By realizing the relationship between albumin and its receptors for the first time, these enabling technologies have the potential to revolutionize the wider healthcare industry by increasing a protein's half-life from minutes to hours, and hours to days.
Mark Perkins is the customer solution manager at Novozymes Biopharma and works with partners who are evaluating Novozymes Biopharma's recombinant albumin products and associated technologies in the areas of biopharmaceutical formulation and half-life extension. He is a formulation chemist with a doctoral degree in pharmaceutical sciences from the University of Nottingham.



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