Nam Ah Kim

Evaluation of taste-masking effects of pharmaceutical sweeteners with an electronic tongue system.

Abstract Electronic tongue systems have been developed for taste measurement of bitter drug substances in accurate taste comparison to development palatable oral formulations. This study was to evaluate the taste masking effect of conventional pharmaceutical sweeteners such as neohesperidin dihydrochalcone, sucrose, sucralose and aspartame. The model drugs were acetaminophen, ibuprofen, tramadol hydrochloride, and sildenafil citrate (all at 20 mM). The degree of bitterness was measured by a multichannel taste sensor system (an electronic tongue). The data was collected by seven sensors and analyzed by a statistical method of principal components analysis (PCA). The effect of taste masking excipient was dependent on the type of model drug. Changing the concentration of taste masking excipients affected the sensitivity of taste masking effect according to the type of drug. As the excipient concentration increased, the effect of taste masking increased. Moreover, most of the sensors showed a concentration-dependent pattern of the taste-masking agents as higher concentration provided higher selectivity. This might indicate that the sensors can detect small concentration changes of a chemical in solution. These results suggest that the taste masking could be evaluated based on the data of the electronic tongue system and that the formulation development process could be performed in a more efficient way.

Comprehensive evaluation of etanercept stability in various concentrations with biophysical assessment

The effect of protein concentration on biophysical stability of etanercept was investigated to monitor its effect on protein formulation development. The conformational and accelerated storage stability of etanercept (marketed as Enbrel(®)) was examined by biophysical analyses including CD, FTIR, DSC, and DLS together with size-exclusion chromatography (SEC). As concentration of etanercept decreased, conformational stability (Tm) decreased with increasing hydrodynamic size and zeta potential. Decreasing secondary structural stability was also observed for relative helix and β-sheet contents. Further investigation examined the accelerated storage stability at different incubation temperatures. Low protein concentration (0.25 and 0.5mg/mL) at 4°C and 30°C exhibited fast monomer loss compared to high concentration (25 and 50mg/mL). The lowest etanercept concentration of 0.25mg/mL displayed the fastest monomer loss and increased fragments since it had lowest Tm values. However, at 50°C, a marked increase in aggregation was observed at high concentrations, as well as accelerated monomer loss into multimers and insoluble aggregates. Induced insoluble aggregation of etanercept was dependent on its concentration and no significant aggregation issues were found at low concentrations such as 0.25 and 0.5mg/mL. The results indicated that the conformational stability of protein solution involved steric repulsion of neighboring protein molecules. Electrostatic circumstances and structural interactions resulted in low stability at low concentrations of etanercept under heat stress. Therefore, it might be recommended to be less diluted during protein formulation development, even in the earlier stages of investigation, to avoid undesirable results.

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.

Glycoengineering of interferon-β 1a improves its biophysical and pharmacokinetic properties.

The purpose of this study was to develop a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a) to improve its biophysical properties, such as aggregation, production and stability, and pharmacokinetic properties without jeopardizing its activity. To achieve this, we introduced additional glycosylation into rhIFN-β 1a via site-directed mutagenesis. Glycoengineering of rhIFN-β 1a resulted in a new molecular entity, termed R27T, which was defined as a rhIFN-β mutein with two N-glycosylation sites at 80th (original site) and at an additional 25th amino acid due to a mutation of Thr for Arg at position 27th of rhIFN-β 1a. Glycoengineering had no effect on rhIFN-β ligand-receptor binding, as no loss of specific activity was observed. R27T showed improved stability and had a reduced propensity for aggregation and an increased half-life. Therefore, hyperglycosylated rhIFN-β could be a biobetter version of rhIFN-β 1a with a potential for use as a drug against multiple sclerosis.

Fundamental analysis of recombinant human epidermal growth factor in solution with biophysical methods

Abstract Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (Tm), intermolecular β-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since Tm increased and β-sheet contents decreased. The contents of induced intermolecular β-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where Tm of first transition was all above 70 °C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.

Biophysical stability of hyFc fusion protein with regards to buffers and various excipients.

A novel non-cytolytic hybrid Fc (hyFc) with an intact Ig structure without any mutation in the hyFc region, was developed to construct a long-acting agonistic protein. The stability of interleukin-7 (IL-7) fused with the hyFc (GXN-04) was evaluated to develop early formulations. Various biophysical methods were utilized and three different buffer systems with various pH ranges were investigated including histidine-acetate, sodium citrate, and tris buffers. Various excipients were incorporated into the systems to obtain optimum protein stability. Two evident thermal transitions were observed with the unfolding of IL-7 and hyFc. The Tm and ΔH increased with pH, suggesting increased conformational stability. Increased Z-average size with PDI and decreased zeta potential with pH increase, with the exception of tris buffer, showed aggregation issues. Moreover, tris buffer at higher pH showed aggregation peaks from DLS. Non-ionic surfactants were effective against agitation by outcompeting protein molecules for hydrophobic surfaces. Sucrose and sorbitol accelerated protein aggregation during agitation, but exhibited a protective effect against oxidation, with preferential exclusion favoring the compact states of GXN-04. The stability of GXN-04 was varied by basal buffers and excipients, hence the buffers and excipients need to be evaluated carefully to achieve the maximum stability of proteins.

Evaluation of protein formulation and its viscosity with DSC, DLS, and microviscometer

The viscosity of highly concentrated protein solutions was evaluated using lysozyme as model protein. Viscosity profiles of lysozyme were examined with the effect of buffer and pH-value at various concentrations. The viscosity of lysozyme dissolved in water increased continuously with the concentration as the slope of shear stress against shear rate increased with the concentration. In addition, the viscosity of lysozyme was higher in histidine buffer than in acetate buffer at selected pH ranges. The effect of various excipient concentrations was also investigated in means of unfolding transition temperature (Tm), viscosity, hydrodynamic size and zeta potential by using differential scanning calorimetry (DSC), microviscometer and dynamic light scattering (DLS). The selected excipients except surfactants increased the viscosity of protein solution with their concentration. Carbohydrates increased the viscosity relatively higher than amino acids and also they increased the conformational stability (Tm) by enhancing the protein molecule more in compact form. Also amino acids increased the viscosity but decreased the conformational stability since they seemed to be only dispersed in the solution avoiding protein–protein interactions, resulting in a decrease of zeta potential. Consequently, the applied methods—DSC, DLS and microviscometer demonstrated the potential to develop a highly concentrated protein formulation to decrease the high viscosity effect with acceptable conformational stability.

Process cycle development of freeze drying for therapeutic proteins with stability evaluation

Many therapeutic proteins have been launched in market or gone into development stages for their high therapeutic efficacy. The proteins can be developed mainly as liquid or solid dosage forms; pre-filled syringes or freeze-dried. Regardless of the dosage forms, they have several stability issues due to the intrinsic properties of the proteins, which can have adverse effects on their efficacy such as loss of bioactivity and immunogenicity. In order to achieve enough stability of proteins, a solid-state dosage form, freeze-dried, has been preferred as providing a better shelf-life. Freeze drying process has become an important method to manufacture, store, and distribute the protein drug products. Despite its advantages, the freeze drying process still has challenges of stability issues and requires optimization. This review provides a basic concept of the freeze drying process while highlighting several stability issues encountered during the development of freeze drying cycle for protein formulations. Furthermore, various excipients used to stabilize freeze-dried protein formulations are also introduced.

Arginine as a protein stabilizer and destabilizer in liquid formulations.

Even though arginine monohydrochloride (ArgHCl) is a useful additive for protein stabilization, its mechanism is not yet fully elucidated. Moreover, there is a concern that ArgHCl may be a protein denaturant since it decreases transition melting temperature (Tm) of certain proteins. It contains a guanidinium group, a critical structure for denaturating activity of guanidine hydrochloride (GndHCl). Effects of ArgHCl, GndHCl, and creatinine on a model protein, etanercept, were examined by biophysical analyses including DLS, DSC, FT-IR, microviscometer, and SEC. Accelerated storage stability of the protein was examined in the absence and presence of H2O2 at different incubation temperatures with pH monitoring. ArgHCl reduced protein aggregation and retained monomer, but increased fragmentation at high temperature. Tm1 and Tm2 of the protein increased with ArgHCl, but slight decrease (>1°C) in Tm3 was observed. GndHCl and creatinine decreased all three Tms. In the presence of heat and H2O2, the effect of ArgHCl was significantly decreased compared to GndHCl and creatinine. In addition, it accelerated the loss of monomer and increased fragmentation with decreasing pH. ArgHCl differed from GndHCl in the mode of physical interaction with the protein, due to its unique balance of three steric functional groups (guanidinium, carboxylic acid, and carbon aliphatic straight chain). In contrast, ArgHCl acted as a protein denaturant at high temperature since NOx generated from the amine group at the 3-carbon aliphatic straight chain and it is supported by GndHCl which did not change the pH nor accelerated the monomer loss after oxidation by H2O2 at high temperature.

Evaluation of etanercept degradation under oxidative stress and potential protective effects of various amino acids.

To evaluate the oxidative stability of proteins, a model protein, etanercept, was exposed to oxidative stress conditions using hydrogen peroxide. Various amino acids were also evaluated on their antioxidant effect. Transition temperature (Tm), secondary structural content, hydrodynamic size, and aggregation and fragmentation of etanercept in solution were assessed using dynamic light scattering (DLS), size exclusion chromatography (SEC), differential scanning calorimetry (DSC), and far-UV circular dichroism (CD). Sample solutions were stored at 4 °C, 20 °C, and 40 °C under oxidative stress. The DLS results exhibited a decrease in the Z-average and intensity peak size of etanercept during the storage, suggesting fragmentation issues rather than aggregation by oxidation. The SEC results exhibited an increase in fragmentation and a decrease in aggregation and monomer content. The monomer content remained higher in histidine than in other amino acids, followed by methionine. There were three Tm of etanercept that were selected as key parameters of conformational stability. Oxidized samples exhibited a significant decrease in Tm values, indicating decreased conformational stability. Methionine exhibited the highest values in Tm1, followed by histidine. The CD spectrum exhibited one unique negative peak of etanercept without amino acids, and changed with oxidation. Only methionine exhibited an enhancement of the stability. All four biophysical analyses results suggest that the histidine and methionine provide a protective effect in the protein solution against oxidative stress. However, histidine was effective as an antioxidant but methionine showed highly enhanced conformational and secondary structural stability.