Jun Yeul Lim

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.

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.

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.

A Novel Experimental Design Method to Optimize Hydrophilic Matrix Formulations with Drug Release Profiles and Mechanical Properties

To investigate the effects of hydrophilic polymers on the matrix system, an experimental design method was developed to integrate response surface methodology and the time series modeling. Moreover, the relationships among polymers on the matrix system were studied with the evaluation of physical properties including water uptake, mass loss, diffusion, and gelling index. A mixture simplex lattice design was proposed while considering eight input control factors: Polyethylene glycol 6000 (x1 ), polyethylene oxide (PEO) N-10 (x2 ), PEO 301 (x3 ), PEO coagulant (x4 ), PEO 303 (x5 ), hydroxypropyl methylcellulose (HPMC) 100SR (x6 ), HPMC 4000SR (x7 ), and HPMC 10(5) SR (x8 ). With the modeling, optimal formulations were obtained depending on the four types of targets. The optimal formulations showed the four significant factors (x1 , x2 , x3 , and x8 ) and other four input factors (x4 , x5 , x6 , and x7 ) were not significant based on drug release profiles. Moreover, the optimization results were analyzed with estimated values, targets values, absolute biases, and relative biases based on observed times for the drug release rates with four different targets. The result showed that optimal solutions and target values had consistent patterns with small biases. On the basis of the physical properties of the optimal solutions, the type and ratio of the hydrophilic polymer and the relationships between polymers significantly influenced the physical properties of the system and drug release. This experimental design method is very useful in formulating a matrix system with optimal drug release. Moreover, it can distinctly confirm the relationships between excipients and the effects on the system with extensive and intensive evaluations.

Effects of annealing on the physical properties of therapeutic proteins during freeze drying process

Effects of annealing steps during the freeze drying process on etanercept, model protein, were evaluated using various analytical methods. The annealing was introduced in three different ways depending on time and temperature. Residual water contents of dried cakes varied from 2.91% to 6.39% and decreased when the annealing step was adopted, suggesting that they are directly affected by the freeze drying methods Moreover, the samples were more homogenous when annealing was adopted. Transition temperatures of the excipients (sucrose, mannitol, and glycine) were dependent on the freeze drying steps. Size exclusion chromatography showed that monomer contents were high when annealing was adopted and also they decreased less after thermal storage at 60°C. Dynamic light scattering results exhibited that annealing can be helpful in inhibiting aggregation and that thermal storage of freeze-dried samples preferably induced fragmentation over aggregation. Shift of circular dichroism spectrum and of the contents of etanercept secondary structure was observed with different freeze drying steps and thermal storage conditions. All analytical results suggest that the physicochemical properties of etanercept formulation can differ in response to different freeze drying steps and that annealing is beneficial for maintaining stability of protein and reducing the time of freeze drying process.

Chemical stability and in vitro and clinical efficacy of a novel hybrid retinoid derivative, bis-retinamido methylpentane.

The anti-aging agent, retinol, has fewer side effects and similar biological activity compared to retinoic acid. However, retinol becomes unstable when exposed to light and heat. A novel hybrid retinoid derivative, bis-retinamido methylpentane (RS-2A), was newly developed to overcome the limitations. This study evaluated the chemical stability of RS-2A under thermal and light conditions by examining degradation profiles, and assessed the in vitro biological activity, cytotoxicity, and clinical efficacy. Chemical stability and degradation profiles were investigated with HPLC and LC-MS. Especially, photo-stability of RS-2A was analyzed under various conditions, such as change of physical state and concentration, different solvents, and various excipients. For analyses of cellular activity and cytotoxicity, human dermal fibroblasts were cultured with RS-2A. To evaluate the safety and efficacy of the compound with the cellular results, RS-2A was applied to women who had moderate to severe wrinkles at the periorbital region. All of the experiments were conducted with retinol as a reference. RS-2A was more stable than retinol to thermal conditions, especially in solution. Both RS-2A and retinol were unstable to light, but RS-2A showed enhanced photo-stability with regard to concentration, more polar solvent, and addition of proper excipients. RS-2A exhibited decreased cytotoxicity and enhanced effects on collagen synthesis compared with retinol. In a clinical study, a 4-week treatment with RS-2A significantly improved the appearance of periorbital wrinkles without any side effects. The results indicate that RS-2A might have potential as an anti-aging agent for cosmeceutical preparations because of its enhanced chemical stability, biological activity, safety, and clinical efficacy.