by Michelle Amaral, Science Writer
This article, written by our Science Writer/PR Consultant, was published in the September 2013 issue of ON drugDelivery Magazine. We are excited that this issue will be circulated at the 3rd Annual PODD – Partnership Opportunities in Drug Delivery – Conference in Boston and at the CPhI Worldwide Pharma Expo CPhI Worldwide Pharma Expo
In this piece, we summarize some of the techniques available for the generation of different formulations of recombinant protein therapeutics, and high-throughput technologies that reduce the time required to screen for the optimal formulation.
A revolutionary moment for the pharmaceutical industry occurred with the advent of recombinant technologies, which enabled proteins to be manufactured and then delivered to humans or animals for the purpose of treating illness and disease. As a result, a wide range of therapeutic approaches opened up: aberrant proteins causing a disease state could be replaced; novel proteins combatting a particular disease could be introduced; and delivery of small-molecule pharmaceuticals could be enhanced through conjugation to a protein or antibody.
In the late 1970s, insulin was the first human protein to be produced using genetic engineering technologies and became commercially available in 1982, greatly advancing the treatment of diabetes. However, the use of proteins in therapeutic strategies presents a number of challenges, namely the requirement that a protein be highly concentrated, stable, and active in solution when delivered.
Fresh, innovative approaches are now providing valuable solutions for protein pharmaceuticals. The aforementioned challenges can be confronted at various points along the path of protein production. In the early stages of development, a protein’s solubility can be affected by the choice of expression vector and the option of adding a fusion tag to the protein of interest. Further downstream, self-interaction chromatography (SIC) is being used as a fast and effective method for determining the most optimal formulation of a protein solution.
The selection of a high-performance expression vector is a must when creating a strategy for recombinant protein production, as it can play an integral role in promoting a soluble product in addition to an optimal yield. In many cases, these vectors encode for extra amino acids that are then attached, or fused, to the prtotein of interest upon expression. The result is a fusion protein with greater solubility than that of the native macromolecule expressed alone. Oftentimes, the fusion partner even contains a region of histidine “tags” that enable quick, efficient purification with affinity chromatography and a cleavage site that allows its subsequent removal using a specific protease treatment.
A fusion partner can range in size from a few amino acids to approximately 25 kDa. The added residues create disorder, allowing greater distance between each protein molecule and thus giving the protein of interest “space” to fold properly. This process enhances solubility of the target protein and prevents aggregation. Activity assays have been performed on numerous representative proteins, demonstrating that functionality is preserved.
Protein solubility can also be enhanced after its production by formulating a buffer solution with conditions that are optimal for proper folding and stability. This is an important step along the light scattering effects of some additives. SIC requires a small amount of protein that is covalently attached to beads and packed into a microcapillary HPLC column. The mobile phase of the SIC experiments consists of the formulation being tested, along with a 1uL bolus injection of the protein of interest. The elution of the protein injected in the mobile phase is measured via UV detection and the retention time is used to evaluate whether or not the formulation or additive of interest is causing attraction or repulsion between the protein molecules.
Data collected via SIC enables the calculation of a parameter that quantitatively describes the protein’s interaction with itself; this is the second virial coefficient, or B value. The B value is the sum of all potential forces between two proteins including ionic, dipole, hydrophobic, and van der Waals forces; it is a measure of protein flexibility in all orientations and distances. In general, positive B values indicate a net repulsion between two protein molecules while negative B values indicate a net attraction. When additives are introduced into solutions, the B value is altered such that the protein molecules display mild attraction to each other, which is conducive to crystallization, or enhanced repulsion, which increases the protein’s physical stability and solubility.
Experimentally determined B values can be used to predict the B value of a protein in over 12,000 other formulations using an artificial neural network. This is a wealth of information for groups that are developing a new biopharmaceutical. Differential scanning calorimetry and other biophysical techniques are performed to confirm a complete characterization profile on the final solutions.
Treatments for disease have drastically improved since the advent of protein therapeutics. In order to be successful, though, a protein must be highly concentrated, stable, and active in solution without aggregation or other phase changes that are detrimental to the drug delivery process and the patient. Solubility can be enhanced at several points along the protein production pipeline. Creating disorder by fusing extra amino acid residues to the protein of interest generates distance between each molecule, enhancing the ability of the protein to fold correctly. An optimal formulation for the protein of interest is crucial for protein performance as well.
High-throughput methods that screen components of the solution save time and money for a company developing a promising drug.