Protein drugs are the fastest growing sector of the biopharmaceutical industry. While usually administered as solutions, many protein drugs are sold as amorphous solid powders, a form often chosen to preserve stability and prolong shelf-life. Proteins can still degrade in the solid state, however. Arguably, the most serious type of degradation is aggregation. The presence of aggregates is associated with changes in drug potency, which may be either greater or less than in the aggregate-free form. Aggregates are also associated with an increased potential for adverse immune responses in patients, which can be life-threatening. As a result, aggregates must be detected and removed during the manufacture of protein drugs. This adds to their cost, a burden ultimately borne by the public. The goal of this ongoing research program is to develop an efficient, designed approach to preventing protein aggregation in amorphous solids based on a thorough understanding of the mechanisms involved. The central hypothesis is that protein aggregation in amorphous solids is the result of specific chemical reactions and changes in protein structure that can be defined with high resolution and prevented by designing the solid environment.
Specific Aim 1 will assess the effects of protein structure on thiol-disulfide exchange in amorphous solids, one of the most common routes to covalent aggregation. The studies test the hypothesis that the rates and mechanisms of thiol-disulfide exchange are affected by protein structure and differ in solution and in amorphous solids.
Specific Aim 2 will develop solid-state photolytic labeling (ssPLL) and solid- state hydrogen deuterium exchange (ssHDX) to map protein-matrix interactions in amorphous solids with high resolution. The studies test the hypothesis that ssHDX and ssPLL are better indicators of aggregation propensity in amorphous solids than current measures of protein structure and solid properties.
Specific Aim 3 will create artificial chaperones that inhibit the aggregation of IgG antibodies. The studies test the hypothesis that solid formulations containing artificial chaperones show greater inhibition of IgG aggregation than controls with common additives. The work is relevant to the NIH mission of advancing the Nation's capacity to protect and improve health in that it addresses methods to preserve the potency and safety of a rapidly growing class of drugs. The work is also consistent with the agency's goal of ensuring a continued high return on the public investment in research by providing tools and knowledge for developing active proteins into marketable drug products.

Public Health Relevance

Aggregates are an impurity in protein drug products that increase the potential for life-threatening immunogenic reactions in patients. Understanding and preventing aggregate formation in amorphous solids will help ensure the safety of this rapidly growing class of drugs. The work will also help to control drug development costs by providing a rational basis for formulation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biomaterials and Biointerfaces Study Section (BMBI)
Program Officer
Smith, Ward
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Purdue University
Schools of Pharmacy
West Lafayette
United States
Zip Code
Chandrasekhar, Saradha; Epling, Daniel E; Sophocleous, Andreas M et al. (2014) Thiol-disulfide exchange in peptides derived from human growth hormone. J Pharm Sci 103:1032-42
Topp, Elizabeth M (2014) Commentary: current perspectives on the aggregation of protein drugs. AAPS J 16:413-4
Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G et al. (2014) Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS). Mol Pharm 11:1869-79
Iyer, Lavanya K; Moorthy, Balakrishnan S; Topp, Elizabeth M (2013) Photolytic labeling to probe molecular interactions in lyophilized powders. Mol Pharm 10:4629-39
Sophocleous, Andreas M; Topp, Elizabeth M (2012) Localized hydration in lyophilized myoglobin by hydrogen-deuterium exchange mass spectrometry. 2. Exchange kinetics. Mol Pharm 9:727-33
Sophocleous, Andreas M; Zhang, Jun; Topp, Elizabeth M (2012) Localized hydration in lyophilized myoglobin by hydrogen-deuterium exchange mass spectrometry. 1. Exchange mapping. Mol Pharm 9:718-26
Zhang, Jun; Topp, Elizabeth M (2012) Protein G, protein A and protein A-derived peptides inhibit the agitation induced aggregation of IgG. Mol Pharm 9:622-8
Thing, Mette; Zhang, Jun; Laurence, Jennifer et al. (2010) Thiol-disulfide interchange in the tocinoic acid/glutathione system during freezing and drying. J Pharm Sci 99:4849-56