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" \n\nABSTRACT\n\n  \n\nFormulation of pharmaceuticals to deliver safe, stable active drug products is crucial to both patient and manufacturer. Multifunctional excipients, such as albumin, incorporated into the formulation process facilitate the stabilization of the drug product from degradation and assist in the administration and release of the active component.\n\n \n\nThe ability of excipients to stabilize and protect an active pharmaceutical ingredient (API) against degradation has caused excipient selection to become more important early in the drug-development process. During manufacturing, transport, and storage, the API can be exposed to a variety of stresses (e.g., temperature variation, pH changes, shear stresses, and freezing) that promote protein degradation, such as denaturation and aggregation. With the potential to increase the immunogenicity and decrease the efficacy and shelf life of the protein drug product, protein stability is a key issue in the final product. Investigating excipient-drug interaction during preformulation stages means that excipient selection, which achieves the desired properties for the final drug product, can be thoroughly explored (1).   \n\n \n\nIncreasing regulatory pressure on the safety, purity, and standardization of excipients has seen the formation of the International Pharmaceutical Excipients Council (IPEC), which defines excipients as substances other than the active drug that have been appropriately evaluated for safety and are included in a drug-delivery system to aid processing of the system during manufacture, and enhance any other attribute of the overall safety and effectiveness of the drug product during storage and use. Moreover, concerns from regulatory bodies such as FDA have prompted manufacturers to implement quality-by-design principles, where thorough characterization of all material components in the manufacturing process is preferred (2). \n\n \n\nOften a drug product will require multiple excipients to obtain optimal stability, quality, and efficacy. In each case, the excipient must be tested for drug-excipient interactions that may potentially alter the drug and excipient functionality. In addition, formulation of challenging molecules such as complex small molecules, macromolecules, and peptides may not be achieved with conventional stabilizing excipients such as sugars, amino acids, and detergents (SADs). It is therefore crucial that excipient manufacturers work closely with the formulators and provide full characterization and understanding of excipients to be used in a drug formulation (1, 3). This article describes a recombinant human albumin (rAlbumin), as an example of a fully characterized multifunctional excipient for the formulation of protein therapeutics and explores its ability to prevent or minimize physical and chemical degradation of drug substances in various test formulations.  \n\n   \n\n \n\nOXIDATION\n\n  \n\nOxidation of drug substances is a common pathway for drug degradation both in liquid and solid formulation, particularly during storage. Factors promoting the oxidation rate of proteins during storage include oxygen (from head space), light, peroxides, and heavy metal ions. Modifications to protein therapeutics through oxidation can lead to a range of functional consequences, such as inactivation or activation of the drug product, altered binding activities, increased susceptibility to aggregation and proteolysis, and altered immunogenicity. Methionine residues on the surface of the protein are the most susceptible to oxidation, and formulation excipients are often used to protect the protein from the oxidation process. Moreover, FDA guidelines suggest that oxidation must be controlled in the product formulation of therapeutic proteins (4). \n\n \n\nTo examine the ability of rAlbumin to act as an antioxidant, pharmaceutically relevant concentrations of  Insulin-like growth factor-I (IGF-I) were exposed to trace amounts of the oxidizing agent hydrogen peroxide (H2O2). IGF-I, an important anabolic growth factor, has been shown to be susceptible to oxidation, particularly during storage, and was therefore chosen as an appropriate model to investigate the ability of rAlbumin as an excipient to inhibit oxidation (5).  \n\n \n\nrAlbumin and L-methionine, a commonly used antioxidant, were tested at a range of concentrations for their ability to prevent oxidation of IGF-I. Either rAlbumin or L-Methionine was dissolved in a buffer solution. The IGF-I (20 μg/ml) protein was then added to all samples followed by the H2O2 to a final concentration of 0.0005%. The reaction was terminated with catalase and the degree of oxidation analyzed by reverse-phase high performance liquid chromatography (HPLC). Percent oxidation of IGF-I was calculated against the main IGF-I peak for all samples.  \n\n\n\nFigure 1 \n\n \n\nResults displayed in Figure 1 demonstrate rAlbumin to be an effective antioxidant. Oxidation of IGF-I was significantly reduced in the solutions containing increasing concentrations of rAlbumin, with the highest concentration (20 mg/ml) reducing IGF-I oxidation by 93%. Due to the susceptibility of IGF-I to oxidation during storage, it is important to note that the initial IGF-I sample already contained 11.6% of the oxidized form. When compared with the commonly used antioxidant L-Methionine, rAlbumin protected IGF-I at molar concentrations approximately 13-fold less than that of L-methionine (see Figure 2).   \n\n\n\nFigure 2 \n\n \n\nHuman serum albumin (HSA) functions as a potent antioxidant primarily due to a single free-thiol at position Cys 34, with HSA-SH acting as a potential scavenger for reactive oxygen species (ROS). The albumin molecule has six methionine residues available as an ROS scavenging system and, in addition, has the ability to bind heavy metal ions, such as Cu2+  and Fe3+, which reduces the availability of these ions to cause oxidation. \n\n   \n\n \n\nAGGREGATION\n\n  \n\nAggregation of protein products is a major concern during the manufacturing and delivery of protein therapeutics.  This concern is primarily because of the increased potential for aggregates to lead to an immunogenic reaction, and possible problems with drug administration (6). There are various mechanisms by which aggregation of proteins occur, including a formation of polymeric-like structures, misfolded proteins, and covalently linked proteins either in a native or denatured state. \n\n \n\nSignificant product losses during manufacture and storage from aggregation are also a concern, influencing product recovery and effective dosage forms. Protein aggregation can occur during numerous stages of the manufacturing and storage process, such as refolding, purification, mixing, freeze-thawing, freeze-drying, and reconstitution. Formation of these associated species is generally concentration dependent, which is a particular challenge for protein therapeutics formulated at high concentrations. \n\n \n\nExposure of protein therapeutics to bulk freeze-thaw processes is a stress that protein drug substances can be exposed to during the manufacturing process to enhance operational flexibility while maintaining product stability. In this study, a range of rAlbumin concentrations was evaluated for the ability to suppress amyloid-fibril formation of the malarial vaccine antigen, merozoite surface protein 2 (MSP-2), after a single freeze-thaw cycle. MSP-2 was chosen as the model to investigate aggregation because of its tendency to form amyloid-like fibril aggregates (7, 8).  \n\n \n\nBriefly, rAlbumin at various concentrations was dissolved in a buffer solution; the MSP-2 protein (3.5 mg/ml) was then added to all samples followed by a single freeze-thaw cycle. Samples were then plated in a 96-well plate and stored at 2–8 °C. Amyloid-like fibrils are known to affect light scattering when measured at λ 320 nm. Therefore, absorbance readings were taken at λ 320 nm at multiple time intervals over a five-day period to test for the formation of aggregation products. \n\n \n\nExcipients commonly used to improve protein stability were also compared against rAlbumin for their ability to inhibit protein aggregation. rAlbumin (15.0 mg/ml), glycine (20.0 mg/ml), PEG 400 (1.0 mg/ml), polysorbate 80 (0.82 mg/ml), or polysorbate 80 (8.2 mg/ml) were tested in the same model as described above. Absorbance readings were taken at multiple time intervals at λ 320 nm.  \n\n\n\nFigure 3 \n\n \n\nResults showed aggregation was suppressed by 50% at a 1:1 molar ratio of the MSP-2 antigen to rAlbumin and at the highest concentration of rAlbumin, aggregation of the MSP-2 protein was reduced by 80% (see Figure 3). These results also demonstrated that rAlbumin suppressed aggregation of the MSP-2 antigen to a greater extent when compared with other commercially available excipients (see Figure 4).  \n\n\n\nFigure 4 \n\n \n\nThe mechanism by which albumin inhibits aggregation is not well understood. HSA is known to sequester and transport metal ions such as Cu and therefore has the potential to reduce the complexes between Cu and amyloid peptides that are involved in the formation of amyloid-like fibrils (9). \n\n   \n\n \n\nADSORPTION\n\n  \n\nLoss of therapeutic proteins through nonspecific adsorption to surfaces during manufacture and storage can be problematic. Nonspecific protein adsorption can lead to structural changes, denaturation, and inactivation of the protein because of irreversible binding events and aggregation. Product loss as a result of these events can significantly decrease the concentration of the active protein in solution, altering the efficacy of the drug. Nonspecific protein adsorption is a particular problem for liquid product at low concentrations. Surfaces to which the protein product is exposed during the manufacturing and storage process which can lead to adsorption include, but are not limited to, silicone tubing, glass and plastic containers, and drug delivery devices. \n\n \n\nProtein adsorption is typically prevented or reduced with the use of a blocking agent through competitive binding of the agent to the surface. Blocking agents commonly used in protein formulation include surfactants such as polysorbate 80 and Tween 20, which bind with higher affinity to surfaces than do proteins or protein-surfactant complexes.  The suitability of these surfactants as protein stabilizers raises certain other concerns because they are potential sources of peroxides, enhancing the likelihood of oxidation of the protein product. \n\n \n\nHSA has also been shown to be an effective blocking agent, allowing manufacturers to prevent thrombosis on plastic surfaces and reduce pressure drop across device modules. The mechanism of action is not well understood, but albumin is thought to bind to charged surfaces through opposite charged functional groups on the molecule. Hydrophobic interactions also occur but at lower strength and are more easily reversed (10).  \n\n \n\nTransforming growth factor-β3 (TGF-β3), an active pharmaceutical ingredient (API) in scarless wound healing, is a hydrophobic protein with a propensity to adsorb to container surfaces. To examine the ability of rAlbumin to act as a suitable blocking agent, the percentage loss of TGF-β3 due to nonspecific binding to polypropylene or glass vial surfaces in the absence and presence of rAlbumin was examined. \n\n \n\nIn this model, TGF-β3 (0.5–60 μg/ml) was added to a polypropylene or glass container containing a buffer solution. Each sample was mixed and centrifuged then transferred to HPLC vials for analysis via reverse-phase HPLC. Percentage recoveries of TGF-β3 were calculated against the TGF-β3 reference standard. rAlbumin was then assessed for its ability to prevent the loss of TGF-β3 to the container surface. rAlbumin (0–0.5 mg/ml), then TGF-β3 (0.2 μg/ml) was added to the test container containing a buffer solution. rAlbumin (0.1 mg/ml) was also compared with polysorbate 80 (0.1 mg/ml) for the ability to protect TGF-β3 (0.2–1.0 μg/ml) against nonspecific adsorption to glass surfaces using the method described above.  \n\n\n\nFigure 5 \n\n \n\nThe results demonstrated rAlbumin significantly reduced protein loss that occurred due to adsorption of TGF-β3 to glass and plastic surfaces. In the absence of rAlbumin, nonspecific binding of TGF-β3 increased as the concentration of TGF-β3 decreased in solution below 60 μg/ml; the recovery of the protein was significantly reduced at lower concentrations (see Figure 5). However, in the presence of rAlbumin the adsorption of TGF-β3 to vessel surfaces was minimal, with greater than 95% recovery achieved using 0.05 mg/ml of rAlbumin (see Figure 6). rAlbumin was then compared to polysorbate 80 in preventing nonspecific binding of proteins to plastic and glass surfaces. In this comparison, rAlbumin prevented the nonspecific binding of TGF-β3 at least as well as polysorbate 80 in plastic containers and significantly better in glass vials.  \n\n\n\nFigure 6 \n\n   \n\n \n\nA MULTIFUNCTIONAL EXCIPIENT\n\n  \n\nSuccessful formulation of therapeutic proteins and peptides that ensures adequate stability, manufacturability, and usability is essential for the biopharmaceutical manufacturer. Excipients are commonly used within the industry to reduce protein degradation, which can occur through physical or chemical pathways. \n\n \n\nIn this study, a recombinant albumin was seen to function as an effective multifunctional excipient to protect proteins against aggregation, especially amyloid-like fibril products, act as an antioxidant in preventing protein oxidation, and as a blocking agent to prevent nonspecific adsorption to surfaces. rAlbumin has the potential to reduce the total number of excipients required, especially for difficult to formulate molecules, simplifying the formulation strategy and accelerating development time in  achieving the desired properties for the finished drug product. \n\n \n\nMark Perkins is a customer solution specialist, Novozymes Biopharma,  mrpk@novozymes.com.  \n\n \n\n \n\nREFERENCES\n\n  \n\n1. Crowley, P.J. and L.G. Martini, Chemistry Today 28 (5), 7–13 (2010).  \n\n \n\n2. F.G. Vogt and A.S. Kord, J. Pharm. Sci. 100 (3). 797–892 (2011). \n\n \n\n3. S.S. Bharate, S.B. Bharate, and A.N. Bajaj, J. Excipients and Food Chemicals, 1 (3),  3–26 (2010). \n\n \n\n4. ICH, Q1A(R2) Stability Testing of New Drug Substances and Products (2003). \n\n \n\n5. J.R. Fransson, J. Pharm. Sci. 86 (9), 1046–1050 (1997). \n\n \n\n6. M.E. Cromwell, M. E. Hilario, and F. Jacobson, The AAPS Journal 8 (3),  572–579 (2006). \n\n \n\n7. W. Wang et al.,  J. Pharm. Sci. 96 (1), 1–6 (2007). \n\n \n\n8. L.A. Kueltzo et al., J. Pharm. Sci. 97 (5), 1801–1812 (2008). \n\n \n\n9. L. Perrone et al., ChemBioChem 11 (1), 110–118 (2010). \n\n \n\n10. Y. Jeyachandran et al., Langmuir 25 (19), 11614–11620 (2009). "
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