A Comprehensive Assessment of All-Oleate Polysorbate 80: Free Fatty Acid Particle Formation, Interfacial Protection and Oxidative Degradation.

PURPOSE: Enzymatic polysorbate (PS) degradation and resulting free fatty acid (FFA) particles are detrimental to biopharmaceutical drug product (DP) stability. Different types and grades of polysorbate have varying propensity to form FFA particles. This work evaluates the homogenous all-oleate (AO) PS80 alongside heterogeneous PS20 and PS80 grades in terms its propensity to form FFA particles and other important attributes like interfacial protection and oxidation susceptibility. METHODS: FFA particle formation rates were compared by degrading PS using non-immobilized hydrolases and fast degrading DP formulations. Interfacial protection of monoclonal antibodies (mAbs) was assessed by agitation studies in saline using non-degraded and degraded PS. Several antioxidants were assessed for their ability to mitigate AO PS80 oxidation and subsequent mAb oxidation by a 40°C placebo stability study and a 2, 2'-Azobis (2-amidinopropane) dihydrochloride stress model, respectively. RESULTS: Visible and subvisible particles were significantly delayed in AO PS80 formulations compared with heterogeneous PS20 and PS80 formulations. Non-degraded AO PS80 was less protective of mAbs against the air-water interface compared with heterogeneous PS20. Interfacial protection by AO PS80 improved upon degradation owing to high surface activity of FFAs. Diethylenetriaminepentaacetic acid (DTPA) completely mitigated AO PS80 oxidation unlike L-methionine and N-Acetyl-DL-Tryptophan. However, DTPA did not mitigate radical mediated mAb oxidation. CONCLUSION: AO PS80 is a promising alternative to reduce FFA particle formation compared with other PS types and grades. However, limitations observed here---such as lower protection against interfacial stresses and higher propensity for oxidation---need to be considered in assessing the risk/benefit ratio in using AO PS80.

A Rapid High-Sensitivity Reversed-Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics.

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.

Experimental ocular toxoplasmosis in genetically susceptible and resistant mice.

Genetic factors determining the pathogenesis and course of ocular toxoplasmosis are poorly understood. In this study, we explored the development of experimental ocular pathogenesis in genetically dissimilar mice infected with either the RH strain, the PLK strain, or the immunodominant surface antigen 1 (SAG1 [P30])-deficient mutant of the RH strain of Toxoplasma gondii. At 11 days postinfection, ocular infection of C57BL/6 mice with all of the strains of parasites resulted in severe inflammatory lesions and high numbers of parasites in eye tissue; less severe ocular lesions at earlier histopathology and prolonged survival were observed in this mouse strain infected with either the major surface antigen 1-deficient SAG1(-/-) strain or the less virulent PLK strain compared with RH infection. In contrast, both BALB/c and CBA/J mice had less severe lesions and low numbers of parasites in their eye tissue, and infection developed into the chronic stage in these mice. There were significantly higher serum levels of gamma interferon and tumor necrosis factor alpha in C57BL/6 mice than in BALB/c and CBA/J mice following ocular infection. These observations confirm earlier reports on systemic immunity to these parasites that the route of Toxoplasma infection markedly influences survival of mice. Our data indicate that genetic factors of the host as well as the parasite strain are critical in determining susceptibility to experimental ocular toxoplasmosis in murine models.

Genetic difference in susceptibility to the blood-retina barrier breakdown in diabetes and oxygen-induced retinopathy.

The breakdown of the blood-retina barrier (BRB) is a common feature of diabetic retinopathy. The purpose of the present study is to determine whether there are genetic differences in susceptibility to the breakdown of the BRB in diabetic retinopathy using two rat models. In streptozotocin (STZ)-induced diabetes, Brown Norway (BN) rats developed sustained vascular hyperpermeability in the retina during the entire experimental period (16 weeks of diabetes), while diabetic Sprague Dawley (SD) rats only showed retinal hyperpermeability from 3 to 10 days after the onset of diabetes. The strain difference in permeability was not correlated with the blood glucose levels in these two strains. In oxygen-induced retinopathy (OIR), BN rats developed retinal vascular hyperpermeability from postnatal day 12 (P12) to P22 with a peak at P16, which was 8.7-fold higher than that in the age-matched normal controls. In OIR-SD rats, however, hyperpermeability was observed from P14 to P18, with a peak only 2.2-fold higher than that in the controls. The strain difference in vascular hyperpermeability was correlated with the different overexpression of vascular endothelial growth factor (VEGF) in the retina of these two models. This finding suggests that genetic backgrounds contribute to the susceptibility to diabetic retinopathy.

Vascular endothelial growth factor is increased in aqueous humor of glaucomatous eyes.

PURPOSE: To assess the concentrations of vascular endothelial growth factor (VEGF) in aqueous humor in eyes with and without glaucoma. METHODS: Concentrations of VEGF were measured using a sandwich ELISA kit in aqueous humor aspirates taken during anterior segment surgery from 87 patients, of whom 54 had glaucoma (27 primary open-angle glaucoma, 8 angle-closure glaucoma, 16 exfoliative glaucoma) and 33 had cataract only. RESULTS: Vascular endothelial growth factor was detected in all samples. The concentration in eyes with cataract only without glaucoma was 102.4 +/- 29.7 pg/mL (mean +/- SD), which was significantly lower than that from eyes with glaucoma (146.7 +/- 51.8 pg/mL). There were no significant differences between primary open-angle glaucoma (140.4 +/- 51.0 pg/mL), angle-closure glaucoma (142.8 +/- 40.2 pg/mL), and exfoliative glaucoma (158.6 +/- 58.9 pg/mL). An unusually high VEGF concentration was detected in one eye with neovascular glaucoma (759 pg/mL) and two eyes with uveitic glaucoma (322 pg/mL). No effect of age, gender, or previous history of medical, laser, or surgical treatment of the aqueous humor VEGF concentration could be detected ( > 0.05). Aqueous humor and plasma VEGF concentrations were measured and compared in 46 patients. The aqueous humor VEGF concentration (144.2 +/- 107.9 pg/mL) was significantly higher ( < 0.01) than the plasma concentration (79.2 +/- 46.1 pg/mL). No significant correlation was found between aqueous humor and plasma VEGF concentrations. CONCLUSION: Aqueous VEGF concentration is increased in eyes with glaucoma.

CD40/CD154 ligation is required for the development of acute ileitis following oral infection with an intracellular pathogen in mice.

BACKGROUND & AIMS: Acute inflammatory ileitis occurs in C57BL/6 mice after oral infection with Toxoplasma gondii. We evaluated the role of CD40/CD154 interaction in the development of acute ileitis in this experimental model. METHODS: CD154-/- and anti-CD154 antibody-treated mice as well as chimeric mice, either C57BL/6 or CD40-/- reconstituted with bone marrow from C57BL/6 or CD40-/- mice, were orally infected with cysts. Inflammation was assessed by qualitative histologic and phenotypic analysis of the intestinal compartment at day 7 after infection. Intestinal chemokine and cytokine production was assayed by ribonuclease protection assay. RESULTS: CD154-/- and anti-CD154 monoclonal antibody-treated mice failed to develop an acute, lethal ileitis after oral infection and survived. Chimeric mice reconstituted with bone marrow from C57BL/6 mice developed ileitis and died, whereas those recipient mice deficient in CD40 survived. CD40 expression in the intestine after infection was found principally within the B-cell compartment. A modest increase in CD40 expression in both the macrophage and dendritic cell compartments was also observed. Both chemokine and cytokine expression was up-regulated in those recipients of wild-type bone marrow. Impairment of CD40/CD154 interaction abrogated the production of these proinflammatory productions. CONCLUSIONS: CD40/CD154 interaction is essential to the development of inflammation in this pathogen-driven experimental model of acute ileitis.