Enhanced protein aggregation suppressor activity of N-acetyl-l-arginine for agitation-induced aggregation with silicone oil and its impact on innate immune responses.

Previously, N-acetyl-l-arginine (NALA) suppressed the aggregation of intravenous immunoglobulins (IVIG) more effectively and with a minimum decrease in transition temperature (Tm) than arginine monohydrochloride. In this study, we performed a comparative study with etanercept (commercial product: Enbrel®), where 25 mM arginine monohydrochloride (arginine) was added to the prefilled syringe. The biophysical properties were investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow-imaging microscopy (FI). NALA retained the transition temperature of etanercept better than arginine, where arginine significantly reduced the Tm by increasing its concentration. End-over-end rotation was applied to each formulation for 5 days to accelerate protein aggregation and subvisible particle formation. Higher monomeric content was retained with NALA with a decrease in particle level. Higher aggregation onset temperature (Tagg) was detected for etanercept with NALA than arginine. The results of this comparative study were consistent with previous study, suggesting that NALA could be a better excipient for liquid protein formulations. Agitated IVIG and etanercept were injected into C57BL/6J female mice to observe immunogenic response after 24 h. In the presence of silicone oil, NALA dramatically reduced IL-1 expression, implying that decreased aggregation was related to reduced immunogenicity of both etanercept and IVIG.

Off-label use of plastic syringes with silicone oil for intravenous infusion bags of antibodies.

The formation of particulates in post-manufacture biopharmaceuticals continues to be a major concern in medical treatment. This study was designed to evaluate the content of micro-sized particles using flow imaging of antibodies in intravenous infusion bags. Intravenous immunoglobulin (IVIG) and Avastin® were selected as model drugs and plastic syringes with and without silicone oil (SO) were used to transfer the drugs into the bags (0.9% saline or 5% dextrose). Antibodies exposed to SO had significantly increased levels of microparticles in both diluents, suggesting SO accelerates particle formation, especially at a higher antibody concentration. Even before the drop stress, their count exceeded the USP guideline. Dropping the bags in the presence of SO produced larger microparticles. Meanwhile, air bubbles were retained longer in saline suggesting more protein film formation on its air-water interface. Overall, both drugs were conformationally stable and produced less particles in dextrose than in saline.

Evaluation of antioxidants in protein formulation against oxidative stress using various biophysical methods.

To evaluate the biophysical stability of protein against oxidative stress, hydrogen peroxide (H2O2) was used to induce non-site-specific protein oxidation. Various biophysical methods were utilized including RP-HPLC, DSC, DLS, and CD. Lysozyme was chosen as a model protein and three different antioxidants (ascorbic acid, N-acetyl-l-cysteine, and l-methionine) were selected to observe their effect. Significant increase in hydrodynamic size, decrease in α-helix propensity, and increase in ß-sheet content evident with increasing H2O2 concentration and temperature suggested methionine residues as the most probable site of oxidation. Among the three anti-oxidants, methionine proved superior in suppressing protein oxidation with its increasing concentration. Methionine reacted with H2O2 to form methionine sulfoxide, which aided in decreasing the oxidant concentration to react with the protein. The hydrodynamic size of methionine containing protein was retained when incubated at 40°C after 14 days with unchanged transition temperature (Tm). In contrast, RP-HPLC revealed oxidation alterations when the same samples were stored at 40°C, highlighting the significant impact of temperature on kinetics. N-acetyl-l-cysteine and ascorbic acid were relatively less protective. Their hydrodynamic size was increased with decreasing Tm compared to the reference. In summary, methionine was a superior antioxidant, implicating a promising component in the protein formulation for suppressing oxidation.

Evaluation of etanercept stability as exposed to various sugars with biophysical assessment.

Even though sugars have been used widely as additives for protein formulations, their exact mechanisms of protein stabilization and applicability remain still in need of investigation. The main purpose of this study was to evaluate the effects of various sugars on the biophysical stability of etanercept (Enbrel(®)). Six well known sugars including glucose, fructose, maltose, sucrose, trehalose, and raffinose were incorporated into the protein solution with different concentrations. The samples were analyzed with dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and size-exclusion chromatography (SEC). The DLS measurement showed that as the number of simple sugars and solution concentration increased, the hydrodynamic size increased with a decreasing absolute zeta potential. The DSC result provided consistent trends with the DLS data. As the concentration of sugar increased, the protein transition temperature (T(m)) was gradually increased in most of samples. In addition, a non-enzymatic browning reaction (NEB) was observed during heating of the sugar solution. To monitor the storage stability, sample solutions were stored at 4 and 40 °C. At 4 °C, the ratio of monomer, aggregate, and fragment were not significantly changed. However, fragmentation of etanercept was observed in accelerated storage. In addition, fructose and maltose showed a peak shift in the SEC result. Those results suggest that the reducing ability of sugar might be a reason for the different etanercept degradation pathways. Therefore, sugars need to be carefully considered to achieve the maximum efficiency of therapeutic proteins for the development of protein formulations.