Flexing the Rules on Drug Dosing Frequency

By Mark Perkins, Novozymes Biopharma | October 9, 2012

The creation of a tunable half-life technology

Pharmaceutical companies are under ever increasing pressure to develop more novel, tailored drugs, while taking new therapeutics through clinical trials to market faster. As a result, the industry is seeing heavy investment in the development of biological drugs, which take much more targeted approaches to the treatment of diseases. A common challenge when using biological drugs is that they are often hampered by short plasma half-lives which can lead to reduced bioavailability, meaning that the human body clears the drug in a matter of minutes or hours. Consequently, patients with chronic conditions require higher dosages and more frequent administration of medications, resulting in the increased likelihood of side effects, greater healthcare costs and, inevitably, reduced patient compliance.

Over recent years there has been much progress in the industry around developing technologies that can address the challenges and enable the serum half-life of protein-based therapeutics to be extended. Current strategies used are those that increase hydrodynamic volume (PEGylation) or those that use FcRn-mediated recycling (albumin fusions). Although current techniques have been successful in extending serum half-life, the ability to design protein half-life to deliver the required pharmacokinetics has not been possible. In response to the issue, a new half-life extension technology has recently been developed to offer a flexible drug delivery platform based on albumin, a natural non-immunogenic plasma protein with a proven and safe regulatory profile that allows manufacturers to tune protein or peptide half-life to specific medical needs. Through subtle modification of the albumin molecule, the new technology enables researchers to flexibly optimize and manage the pharmacokinetics of a target protein while retaining efficiency.

Innovations in Technology
Serum albumin displays a long half-life of 19 days in humans, in contrast to protein therapeutics, which are usually cleared from the body within a matter of minutes or 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-dependant binding to the FcRn in acidic endosomes. This interaction with the FcRn 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 (Figure 1).

Figure 1: Hypothetical model based on knowledge of IgG recycling. The neonatal Fc receptor (FcRn) functions to protect albumin from degradation resulting in prolonged half-life.

It is the pH-dependent interaction between albumin fusion and FcRn that provides the basis for the latest advancements in albumin fusion technology. Understanding the interaction between albumin and FcRn and the impact on albumin fusion half-life has enabled the engineering of this interaction with the potential to modulate albumin’s half-life. Previous studies, which altered the interaction between IgG and FcRn, have been shown to impact the pharmacokinetics of the IgG.

Further research has identified potential amino acids involved in the binding of albumin to FcRn through the analysis of polymorphisms, cross species binding studies and sequence alignments. Subsequently, numerous albumin variants have been generated with single amino acid substitutions. Binding affinity studies of each albumin variant to FcRn at acidic pH have identified single amino acid positions capable of generating a range of affinity variants with distinct binding differences. Variants have been measured with both increased and decreased receptor binding affinities potentially translating to modulation of albumin half-life.

As a result of the new half-life extension technology’s ability to modulate albumin half-life, researchers are able to control and tune their drug design. Although extension of half-life is generally pursued, in situations where a drug is highly toxic a reduced protein half-life can now also be achieved.

Industry-Changing Advantages
The latest advancements in half-life extension technology have been specifically developed to allow users to flexibly tune the pharmacokinetics of a particular target protein or peptide while retaining efficiency. The technology can increase a protein’s half-life from hours to days, days to weeks. As a result, it can be used to flexibly modulate the half-life of the drug to allow the control of how long the drug stays in the human body, depending on what it is being used for. This flexibility to extend or reduce the half-life of proteins allows drug manufacturers to improve treatment regimes and create novel drugs tailored to the specific needs of patients suffering from chronic or acute conditions, such as diabetes, hemophilia and neutropenia.

As there are still only a small number of biologic drugs on the market, companies are looking to adjust and develop those that are available to them. The technology will allow manufacturers to establish a niche position in the market with more innovative and flexible products. On both a commercial and patient-centric level, the innovation will offer companies a definite competitive advantage due to its ability to improve lives of patients who are suffering from chronic illnesses. The technology could lead to lower and less frequent dosage levels for patients who need to take regular medication, resulting in increased patient compliance and the possibility for patients to administer their own drugs.

Drug Delivery Strategies
Depending on specific drug delivery requirements, both conjugation and fusion technology can be used with the half-life extension technology. Table 1 briefly outlines the main features of conjugation and fusion technology.

Table 1: Comparison of albumin fusion strategies. Conjugations vs Genetic fusion

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, which 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. 

To test that the albumin variants maintain their modified FcRn binding affinity when fused to a protein or peptide, a range of albumin protein fusions have been generated. 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 (Figure 2). All albumin fusions tested showed distinct differences in receptor affinity correlating to their unfused variant. Each fusion demonstrated the same changes in ScRn binding as the control rHSA variant.

Figure 2: Albumin variants selected for their different FcRn receptor affinities were genetically fused to a variety of proteins and peptide. Each fusion was tested was found to maintain similar affinity for FcRn receptor when compared to the unfused variant.

The flexibility of albumin technology enables proteins and peptides to be bound at either the C- or N-terminus or both, generating 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. The technology also enables construction of albumin variants with altered binding affinity to FcRn, making it possible to modulate half-life extension of fused target proteins, offering drug developers enhanced flexibility and control.

During recent years a range of half-life extension platforms have been developed in response to growing market demand for drugs that work more effectively. However, these platforms are often developed using synthetic materials and are unable to increase and decrease the drug half-life according to the needs of a specific medical condition. The latest advancements in half-life extension technology offer a broadly applicable and easily accessed platform that enables drug manufacturers to differentiate their products from competitive therapies by tuning the half-life according to therapeutic requirements. For the first time, the relationship between albumin and its receptor has been resolved, resulting in a new technology that has the potential to revolutionize the healthcare industry. 

Both small and large pharma companies have begun using the technology following its launch to market, particularly in the fields of diabetes, hemophilia and neutropenia. The shift towards the use of this new technology has been driven by the fact that it is based on albumin, a natural non-immunogenic plasma protein that is already naturally present in the human body. As such it offers drug developers a natural, low-cost and safe molecule, in addition to complete control and flexibility of their drug design. By offering the potential to reduce the dosing frequency of a drug from days to weeks, the half-life extension technology can dramatically improve patient quality of life.

Mark Perkins, Ph.D. is a customer solution manager at Novo-zymes Biopharma. He can be reached at mrpk@novozymes.com
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