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The Effect of Adsorption-Induced Conformational Changes on Structure, Activity and Aggregate Formation of Injectable Biologics

i. Protein Adsorption

Proteins are large, complex biomacromolecules containing functional groups of varying size, shape, charge and hydrogen binding capacity. This large variety of functional groups comes from the twenty different amino acid monomers which are linked together in a polypeptide chain to form the protein copolymer. Proteins are intrinsically surface-active because they have amphiphilic characteristics, due to the presence of polar and non-polar side chains creating regions of various hydrophobicity, as well as amphoteric characteristics, due to the presence of acidic and basic side chains resulting in a distribution of surface charge. Because of such chemical and electrical diversity of the protein molecule, and because of the marginal stability of folded proteins in an aqueous environment, spontaneous adsorption will occur in the presence of nearly all surfaces.

ii. Recombinant protein therapeutics and injectables

Presently, more than 130 biologically-active pharmaceuticals, also called biologics, founded on the principles of recombinant DNA protein production, are approved for clinical use by the FDA, and over 200 more are in Phase III clinical trials. Ever since the first recombinant peptide was expressed in E. coli in 1977 and the first FDA-approved genetically engineered protein, insulin, was manufactured by Eli Lilly in 1982, biotechnology products have begun replacing small molecule drugs in the treatment of a wide variety of diseases. Currently, biologics account for nearly $50 billion in annual sales and make up the majority of parenteral drugs in clinical trials. These biologics can be categorized as follows: monoclonal antibodies, recombinant proteins, viral agents, nucleic acids, and bacterial vaccine therapies. Biologics have several advantages over small molecule drugs. Primarily, small molecules simply cannot accomplish the same highly specific and complex set of functions that proteins can. Furthermore, due to the specific action of proteins, there is little chance of protein therapeutics interfering with other, non-related biological activity and cause unknown side-reactions. Biologics also have a decreased level of immunogenicity. Because the body naturally produces many of the proteins used in therapeutics, these molecules are less likely to be recognized by the immune system as antigens. Protein therapeutics are also attractive from a financial perspective: the time required for clinical development and FDA approval is on average one year less for biologics than for small molecules. Moreover, companies are able to obtain far-reaching patents for protein therapeutics. Thus, these factors, as well as the recent advances in protein engineering explain the trend of replacing small molecule drugs by biologically active compounds. While oral administration of medicines is the preferred and most widely used route of administration, this is not feasible for the delivery of biomacromolecules such as proteins, due to their instability in the gastro-intestinal tract and low permeability across the biological membranes lining the capillaries. Thus, parenteral administration is the most common route for biologics. Furthermore, intravenous injection is the most efficient parenteral route of administration for delivering biologics to the systemic circulation. A significant aspect of intravenous injection is that the medicine must be either formulated in the form of a suspension or aqueous solution; or in the case of lyophilized drugs, it must be reconstituted prior to administration.

iii. Protein Stability Challenges

In the production and formulation of biologics, a major challenge and criterion for success is maintaining the physical and chemical stability of the protein throughout its entire lifecycle. Protein structure is flexible and can assume a large number of possible conformations, the relative stabilities of which are sensitive to environmental conditions and external factors such as pH, temperature, ionic strength, presence of ligands, cosolutes or impurities, and surface interactions. Because the therapeutic activity of a protein is highly dependent on its conformation, great care must be taken during the production, storage and handling of the protein to maintain its native structure. Degradation pathways for proteins can be separated into chemical and physical instabilities. Chemical instabilities include deamidation, oxidation, proteolysis, and racemization. Physical instabilities include denaturation, aggregation, surface adsorption, and precipitation. Denaturation refers to the alteration of the tertiary, and sometimes secondary structure, of the protein, which may result in an unfolded state. Between the native and unfolded states, partially unfolded intermediates can occur. Protein adsorption to surfaces can catalyze the structural denaturation of the protein, and may create such partially unfolded intermediates. These intermediates are believed to be the precursors for nonnative aggregate formation, which is an irreversible process that can result in precipitate formation, inhibition of biological activity, and may lead to enhanced immunogenicity. Although physical instabilities are rarely an issue for small molecule drugs; for proteins, physical instability is of great concern for biologics, and will be the main focus of this research project. Current methods employed to improve protein stability in pharmaceutical formulations include chemical modification of the proteins and use of additives, such as excipients or detergents. A commonly used stabilizer in liquid formulations which also prevents adsorption is Human Serum Albumin (HSA). Due to its relatively high affinity to most surfaces, HSA is likely to saturate the surface binding sites and prevent adsorption of the primary protein. However, there are several disadvantages related to the use of HSA. The protein is extracted from human plasma, and carries the risk of introducing blood-borne pathogens into the drug. Recombinant HSA is a potential solution; however it is costly to produce. Additionally, the presence of a secondary protein in a relatively high concentration makes the development of analytical techniques to characterize the primary agent difficult. These reasons support the trend of replacing existing formulas with HSA-free solutions, and understanding and resolving the root causes of protein instability.

iv. Research Aims

Throughout the lifecycle of a protein-based therapeutics, many situations occur where the protein comes into contact with synthetic surfaces. For injectables biologics, during medication storage and administration, the biologically-active molecule, will come into contact with the wall of the storage and delivery device, such as a vial, syringe, intravenous tubing set, or catheter. This protein-surface interaction is even more pronounced when the drug is formulated as a solution, and may be in contact with the storage container for 18-24 months, the average shelf-life of a typical protein pharmaceutical. As previously mentioned, the stability of proteins may be compromised when subjected to liquid/solid interfaces. Therefore, the goal of this research will be to better understand the effect that surfaces used in medication storage and delivery have on the physical stability of biologically-active molecules. Specifically, the impact of surface-induced structural changes on the conformation, activity, and aggregation behavior of protein will be studied, to better understand the negative impact device surfaces may have on the stability of high-value biologics.