Dirk L. Teagarden

Protein aggregation--pathways and influencing factors.

Proteins generally will tend to aggregate under a variety of environmental conditions in comparison with small drug molecules. The extent of aggregation is dependent on many factors that can be broadly classified as intrinsic (primary, secondary, tertiary or quaternary structure) or extrinsic (environment in which protein is present, processing conditions, etc). These protein aggregates may exhibit less desirable characteristics like reduced or no biological activity, potential for immunogenicity or other side effects. Protein aggregation remains one of the major challenges in the development and commercialization of biotechnology products. This article is intended to review and discuss the latest understandings in protein aggregation pathways and the possible extrinsic factors that affect or control the protein aggregation process.

Comparative Evaluation of Disodium Edetate and Diethylenetriaminepentaacetic Acid as Iron Chelators to Prevent Metal -Catalyzed Destabilization of a Therapeutic Monoclonal Antibody

Understanding the effect of metal chelators with respect to their ability to inhibit metal-catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na(2)EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation, and fragmentation demonstrated that DTPA was equivalent to Na(2)EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na(2)EDTA and DTPA exhibited the capacity to reduce protein degradation. However, substoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na(2)EDTA in biologic protein formulations.

Investigation of PEG Crystallization in Frozen and Freeze‐Dried PEGylated Recombinant Human Growth Hormone–Sucrose Systems: Implications on Storage Stability

The objectives of the current study were to investigate (i) the phase behavior of a PEGylated recombinant human growth hormone (PEG-rhGH, ∼60 kDa) during freeze-drying and (ii) its storage stability. The phase transitions during freeze-thawing of an aqueous solution containing PEG-rhGH and sucrose were characterized by differential scanning calorimetry. Finally, PEG-rhGH and sucrose formulations containing low, medium, and high polyethylene glycol (PEG) to sucrose ratios were freeze-dried in dual-chamber syringes and stored at 4°C and 25°C. Chemical decomposition (methionine oxidation and deamidation) and irreversible aggregation were characterized by size-exclusion and ion-exchange chromatography, and tryptic mapping. PEG crystallization was facilitated when it was covalently linked with rhGH. When the solutions were frozen, phase separation into PEG-rich and sucrose-rich phases facilitated PEG crystallization and the freeze-dried cake contained crystalline PEG. Annealing caused PEG crystallization and when coupled with higher drying temperatures, the primary drying time decreased by up to 51%. When the freeze-dried cakes were stored at 4°C, while there was no change in the purity of the PEG-rhGH monomer, deamidation was highest in the formulations with the lowest PEG to sucrose ratio. When stored at 25°C, this composition also showed the most pronounced decrease in monomer purity, the highest level of aggregation, and deamidation. Furthermore, an increase in PEG crystallinity during storage was accompanied by a decrease in PEG-rhGH stability. Interestingly, during storage, there was no change in PEG crystallinity in formulations with medium and high PEG to sucrose ratios. Although PEG crystallization during freeze-drying did not cause protein degradation, crystallization during storage might have influenced protein stability.

Case Study: Parecoxib: A Prodrug of Valdecoxib

Parecoxib, a weakly acidic drug (pKa 4.9), was developed as a COX-2 inhibitor for the management and treatment of acute pain. It was designed to be a water-soluble (> 50 mg/mL in normal saline), parenterally safe prodrug form of valdecoxib, or 4-(5-methyl-3-phenyl-4-isoxazolyl) benzenesulfonamide, a diaryl-substituted isoxazole. Valdecoxib is a sparingly water soluble (10 µ/mL), weakly acidic drug with a pKa of 9.8. Valdecoxib is commercially available as Bextra, an oral formulation for the management of acute pain, chronic pain, osteoarthritis (OA), rheumatoid arthritis (RA), and primary dysmenorrhea.