A comprehensive evaluation of arginine and its derivatives as protein formulation stabilizers.

Arginine and its derivatives (such as arginine ethyl ester and acetyl arginine) have varying degrees of protein aggregation suppressor effect across different protein solutions. To understand this performance ambiguity, we evaluated the activity of arginine, acetyl arginine, and arginine ethyl ester for aggregation suppressor effect against human intravenous immunoglobulin G (IgG) solution at pH 4.8. Both arginine and its cationic derivative arginine ethyl ester in their hydrochloride salt forms significantly reduced the colloidal and conformational stability (reduced kd and Tm) of IgG. Consequently, the monomer content was decreased with an increase in subvisible particulates after agitation or thermal stress. Furthermore, compared to arginine, arginine ethyl ester with one more cationic charge and hydrochloride salt form readily precipitated IgG at temperatures higher than 25 °C. On the contrary, acetyl arginine, which mostly exists in a neutral state at pH 4.8, efficiently suppressed the formation of subvisible particles retaining a high amount of monomer owing to its higher colloidal and conformational stability. Concisely, the charged state of additives significantly impacts protein stability. This study demonstrated that contrary to popular belief, arginine and its derivatives may either enhance or suppress protein aggregation depending on their net charge and concentration.

Optimization of HPLCCAD method for simultaneous analysis of different lipids in lipid nanoparticles with analytical QbD.

Since lipid nanoparticles (LNP) have emerged as a potent drug delivery system, the objective of this study was to develop and optimize a robust high-performance liquid chromatography with charged aerosol detectors (HPLCCAD) method to simultaneously quantify different lipids in LNPs using the analytical quality by design (AQbD) approach. After defining analytical target profile (ATP), critical method attributes (CMAs) were established as a resolution between the closely eluting lipid peaks and the total analysis time. Thus, potential high-risk method parameters were identified through the initial risk assessment. These parameters were screened using Plackett-Burman design, and three critical method parameters (CMPs)-MeOH ratio, flow rate, and column temperature-were selected for further optimization. Box-Behnken design was employed to develop the quadratic models that explain the relationship between the CMPs and CMAs and to determine the optimal operating conditions. Moreover, to ensure the robustness of the developed method, a method operable design region (MODR) was established using the Monte Carlo simulation. The MODR was identified within the probability map, where the risk of failure to achieve the desired CMAs was less than 1%. The optimized method was validated according to the ICH guidelines (linearity: R2 > 0.995, accuracy: 97.15-100.48% recovery, precision: RSD < 5%) and successfully applied for the analysis of the lipid in the LNP samples. The development of the analytical method to quantify the lipids is essential for the formulation development and quality control of LNP-based drugs since the potency of LNPs is significantly dependent on the compositions and contents of the lipids in the formation.

Biophysical characterization of siRNA-loaded lipid nanoparticles with different PEG content in an aqueous system.

Although lipid nanoparticles (LNP) are potential carriers of various pharmaceutical ingredients, further investigation for maintaining their stability under various environmental stressors must be performed. This study evaluated the influence of PEGylation and stress conditions on the stability of siRNA-loaded LNPs with different concentrations of PEG (0.5 mol%; 0.5 % PEG-LNP and 1.0 mol%; 1.0 % PEG-LNP) anchored to their surface. We applied end-over-end agitation, elevated temperature, and repeated freeze and thaw (F/T) cycles as physicochemical stressors of pH and ionic strength. Dynamic light scattering (DLS), flow imaging microscopy (FIM), and ionic-exchange chromatography (IEX) were to determine the degree of aggregation and change in siRNA content. The results indicate that 0.5 % PEG-LNP resisted aggregation only at low pH levels or with salt, whereas 1.0 % PEG-LNP had increased colloidal stability except at pH 4. 0.5 % PEG-LNP withstood aggregation until 71 °C and three cycles of F/T. In contrast, 1.0 % PEG-LNP maintained colloidal stability at 90 °C and seven F/T cycles. Moreover, 1.0 % PEG-LNP had higher siRNA stability under all stress conditions. Therefore, to ensure the stability of LNP and encapsulated siRNA, the PEG concentration must be carefully controlled while considering LNPs' colloidal instability mechanisms under various stress conditions.

Comparative study between a gravity-based and peristaltic pump for intravenous infusion with respect to the generation of proteinaceous microparticles.

Subvisible particles generated during the preparation or administration of biopharmaceuticals might increase the risk of immunogenicity, inflammation, or organ dysfunction. To investigate the impact of an infusion system on the level of subvisible particles, we compared two types of infusion sets based on peristaltic movement (Medifusion DI-2000 pump) and a gravity-based infusion system (Accu-Drip) using intravenous immunoglobulin (IVIG) as a model drug. The peristaltic pump was found to be more susceptible to particle generation compared to the gravity infusion set owing to the stress generated due to constant peristaltic motion. Moreover, the 5-µm in-line filter integrated into the tubing of the gravity-based infusion set further contributed to the reduction of particles mostly in the range ≥ 10 µm. Furthermore, the filter was also able to maintain the particle level even after the pre-exposure of samples to silicone oil-lubricated syringes, drop shock, or agitation. Overall, this study suggests the need for the selection of an appropriate infusion set equipped with an in-line filter based on the sensitivity of the product.

Evaluation of subvisible particles in human immunoglobulin and lipid nanoparticles repackaged from a multi-dose vial using plastic syringes.

The multi-dose vial (MDV) is widely used for most biopharmaceuticals that are repackaged in plastic syringes before use. However, subvisible particle formation with the use of plastic syringes containing silicone oil (SO syringes) for handling therapeutic proteins can be problematic. This study aimed to evaluate the extent of and trends in microparticle (>1 µm) formation and accumulation in repackaged syringes from MDVs containing human immunoglobulin (IgG) and lipid nanoparticles (LNPs). Light obscuration (LO) and flow imaging (FI) were used to analyze the microparticles. The number of microparticles observed with the use SO syringes was greater than that with SO-free syringes, and the number of microparticles continuously increased as did the number of times of repackaging in syringes for both drugs. However, a large variation was observed across different brands of SO syringes. In contrast, using a different technique of drug withdrawal from the vial significantly reduced the number of microparticles. Furthermore, the use of filter-integrated needles or the inclusion of stabilizers such as acetyl-arginine and Tween 20 into the formulation also helped reduce particle formation.

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.

New long-acting injectable microspheres prepared by IVL-DrugFluidic™ system: 1-month and 3-month in vivo drug delivery of leuprolide.

The microspheres for 1-month (PLGA-based) and 3-month (PLA-based) drug releases of leuprolide were manufactured using an IVL-DrugFluidic™ system and their morphology, particle size and distribution, and encapsulation efficiency were compared with the commercialized products. In vivo test was also conducted to monitor the amount of leuprolide and testosterone in plasma after a single subcutaneous injection in male Sprague-Dawley (SD) rats and male Beagle dogs. The median diameter, span value, drug loading, and encapsulation efficiency of PLGA-based microspheres (63.29 µm, 0.26, 13.15%, and 78.90%, respectively) and PLA-based microspheres (80.28 µm, 0.21, 14.42%, and 86.50%, respectively) demonstrated narrow particle size distribution (monodispersed) and efficient drug loading/encapsulation efficiency. Both the microspheres exhibited a desired time-dependent drug release profile and reduced initial burst release by 16-fold in SD rats and 240-fold in Beagle dogs compared to Leuplin DPS®. Moreover, the testosterone level in plasma was suppressed to < 0.50 ng/mL after 28 days with a steady plasma drug concentration. The results suggested that newly developed leuprolide-loaded microspheres produced by the IVL-DrugFluidic™ system could provide extended drug release with advantages such as reduced initial burst release and testosterone level suppression, along with steady plasma drug concentration, over the existing products.

Correlation of Solubility Thermodynamics of Glibenclamide with Recrystallization and In Vitro Release Profile.

The solubility of glibenclamide was evaluated in DMSO, NMP, 1,4-dioxane, PEG 400, Transcutol® HP, water, and aqueous mixtures (T = 293.15~323.15 K). It was then recrystallized to solvate and compressed into tablets, of which 30-day stability and dissolution was studied. It had a higher solubility in 1,4-dioxane, DMSO, NMP (Xexp = 2.30 × 103, 3.08 × 104, 2.90 × 104) at 323.15 K, its mixture (Xexp = 1.93 × 103, 1.89 × 104, 1.58 × 104) at 298.15 K, and 1,4-dioxane (w) + water (1-w) mixture ratio of w = 0.8 (Xexp = 3.74 × 103) at 323.15 K. Modified Apelblat (RMSD ≤ 0.519) and CNIBS/R-K model (RMSD ≤ 0.358) suggested good comparability with the experimental solubility. The minimum value of ΔG° vs ΔH° at 0.70 < x2 < 0.80 suggested higher solubility at that molar concentration. Based on the solubility, it was recrystallized into the solvate, which was granulated and compressed into tablets. Among the studied solvates, the tablets of glibenclamide dioxane solvate had a higher initial (95.51%) and 30-day (93.74%) dissolution compared to glibenclamide reference (28.93%). There was no stability issue even after granulation, drying, or at pH 7.4. Thus, glibenclamide dioxane solvate could be an alternative form to improve the molecule's properties.

Lessons Learned in Protein Precipitation Using a Membrane Emulsification Technique to Produce Reversible and Uniform Microbeads.

The effects of the manufacturing process and the regeneration of Shirasu porous glass (SPG) membranes were investigated on the reproducibility of protein precipitants, termed protein microbeads. Intravenous immunoglobulin (IVIG) was selected as a model protein to produce its microbeads in seven different cases. The results showed that the hydrophobically modified SPG membrane produced finer microbeads than the hydrophilic SPG membrane, but this was inconsistent when using the general regeneration method. Its reproducibility was determined to be mostly dependent on rinsing the SPG membrane prior to the modification and on the protein concentration used for emulsification. The higher concentration could foul and plug the membrane during protein release and thus the membrane must be washed thoroughly before hydrophobic modification. Moreover, the membrane regenerated by silicone resin dissolved in ethanol had better reproducibility than silicone resin dissolved in water. On the other hand, rinsing the protein precipitant with cold ethanol after the emulsification was not favorable and induced protein aggregation. With the addition of trehalose, the purity of the IVIG microbeads was almost the same as before microbeadification. Therefore, the regeneration method, protein concentration, and its stabilizer are key to the success of protein emulsification and precipitation using the SPG membrane.

Three months extended-release microspheres prepared by multi-microchannel microfluidics in beagle dog models.

To evaluate in vivo drug release profiles in beagle dogs, finasteride-loaded PLGA microspheres were prepared using a novel method of IVL-PPF Microsphere® microfluidic device. Briefly, the dispersed phase (PLGA and finasteride in dichloromethane) was mixed with the continuous phase (0.25% w/v PVA aqueous solution) in the parallelized microchannels. After lyophilization, the diameter of the microspheres was around 40 µm (PLGA 7502A or 5002A) and around 30 µm (PLGA/PLA02A mixture). Their CV and span values suggested a narrow size distribution in repeated batch preparations. The in vivo drug release from the PLGA microspheres exhibited three substantial phases: an initial burst, a moderate release, and then a plateau. The microspheres based on PLGA 7502A (75:25 co-polymer) demonstrated extended drug release for around 1 month with a minimized initial burst release compared to PLGA 5002A (50:50 co-polymer). Moreover, the in vivo drug release profile in beagle dogs was proportionally related to the amount of drug loading. Furthermore, the addition of PLA02A into the fabrication of the microsphere synergistically extended the drug release up to 3 months. These results demonstrated the value of this method to achieve uniform microspheres and extend the drug release properties with interpretative in vivo PK profiles.

Protein microbeadification to achieve highly concentrated protein formulation with reversible properties and in vivo pharmacokinetics after reconstitution.

A protein precipitation technique was optimized to produce biophysically stable 'protein microbeads', applicable to highly concentrated protein formulation. Initially, production of BSA microbeads was performed using rapid dehydration by vortexing in organic solvents followed by cold ethanol treatment and a vacuum drying. Out of four solvents, n-octanol produced the most reversible microbeads upon reconstitution. A Shirasu porous glass (SPG) membrane emulsification technique was utilized to enhance the size distribution and manufacturing process of the protein microbeads with a marketized human IgG solution. Process variants such as dehydration time, temperature, excipients, drying conditions, and initial protein concentration were evaluated in terms of the quality of IgG microbeads and their reversibility. The hydrophobized SPG membrane produced a narrow size distribution of the microbeads, which were further enhanced by shorter dehydration time, low temperature, minimized the residual solvents, lower initial protein concentration, and addition of trehalose to the IgG solution. Final reversibility of the IgG microbeads with trehalose was over 99% at both low and high protein concentrations. Moreover, the formulation was highly stable under repeated mechanical shocks and at an elevated temperature compared to its liquid state. Its in vivo pharmacokinetic profiles in rats were consistent before and after the 'microbeadification'.

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.

Mimicking Low pH Virus Inactivation Used in Antibody Manufacturing Processes: Effect of Processing Conditions and Biophysical Properties on Antibody Aggregation and Particle Formation.

Low pH virus inactivation (VI) step is routinely used in antibody production manufacturing. In this work, a mimic of the VI step was developed to focus on evaluating adverse effects on product quality. A commercially available lab-scale glass reactor system was utilized to assess impacts of process and solution conditions on process-induced monoclonal antibody particle formation. Flow imaging was found to be more sensitive than light obscuration in detecting microparticles. NaOH as a base titrant increased protein microparticles more than Tris. Both stirring and NaCl accelerated particle formation, indicating that interfacial stress and protein colloidal stability were important factors. Polysorbate 80 was effective at suppressing particle formation induced by stirring. In contrast, trehalose led to higher microparticle levels suggesting a conformational stabilizer may have other adverse effects during titration with stirring. Additionally, conformational and colloidal stability of antibodies were characterized to investigate the potential roles of antibody physicochemical properties in microparticle formation during VI. The stability data were supportive in rationalizing particle formation behaviors, but they were not predictive of particle formation during the mimicked viral inactivation steps. Overall, the results demonstrate the value of testing various solution and processing conditions in a scaled-down system prior to larger-scale VI bioprocesses.

3D-printed tablets using a single-step hot-melt pneumatic process for poorly soluble drugs.

Main purpose was to evaluate the applicability of a 3D-printer equipped with a hot-melt pneumatic dispenser as a single-step process to prepare tablet dosage forms. Dutasteride, a poorly water-soluble drug, was selected as a model drug. Soluplus®, Kollidon® VA 64, Eudragit® E PO, and hydroxypropyl cellulose (HPC) were premixed as bulking agents prior to printing. Differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and thermogravimetric analysis (TGA) were utilized to evaluate the physicochemical properties of the 3D-printed tablets. Moreover, different geometries were designed to correlate the surface area/volume (SA/V) of the tablets with respect to their release profiles. As a result, printed dutasteride was confirmed to be in an amorphous state and not recrystallized even after the accelerated storage stability. Out of the four bulking agents, Kollidon® VA 64, enhanced the dissolution of the printed dutasteride, reaching above 80% within 15 min. These results suggest that the hot-melt pneumatic dispenser was efficient in converting the solid state into an amorphous state, which significantly enhanced the dissolution. On the other hand, the tube-shaped 3D-printed tablet exhibited the fastest drug dissolution profile, which had the highest SA/V ratio in comparison to the cube, hemisphere, and pyramid shapes. These results confirm the dependency of the drug dissolution rate not only on its crystallinity but also on the surface area of the 3D-printed tablet. Therefore, a 3D-printer equipped with a hot-melt pneumatic dispenser possesses useful applicability in enhancing drug dissolution, especially for poorly water-soluble drugs, in a single-step process.

Solubility Determination of c-Met Inhibitor in Solvent Mixtures and Mathematical Modeling to Develop Nanosuspension Formulation.

The solubility and dissolution thermodynamics of new c-Met inhibitor, ABN401, were determined in eleven solvents and Transcutol® HP-water mixture (TWM) from 298.15 to 318.15 K. The experimental solubilities were validated using five mathematical models, namely modified Apelblat, van't Hoff, Buchowski-Ksiazaczak λh, Yalkowsky, and Jouyban-Acree van't Hoff models. The experimental results were correlated and utilized further to investigate the feasibility of nanosuspension formation using liquid anti-solvent precipitation. Thermodynamic solubility of ABN401 increased significantly with the increase in temperature and maximum solubility was obtained with Transcutol® HP while low solubility in was obtained water. An activity coefficient study indicated that high molecular interaction was observed in ABN401-Transcutol® HP (THP). The solubility increased proportionately as the mole fraction of Transcutol® HP increased in TWM, which was also supported by a solvent effect study. The result suggested endothermic and entropy-driven dissolution. Based on the solubility, nanosuspension was designed with Transcutol® HP as solvent, and water as anti-solvent. The mean particle size of nanosuspension decreased to 43.05 nm when the mole fraction of ABN401 in THP, and mole fraction of ABN401 in TWM mixture were decreased to 0.04 and 0.1. The ultrasonicated nanosuspension appeared to give comparatively higher dissolution than micronized nanosuspension and provide a candidate formulation for in vivo purposes.

N-Acetylated-L-arginine (NALA) is an enhanced protein aggregation suppressor under interfacial stresses and elevated temperature for protein liquid formulations.

Even though arginine hydrochloride has been recognized as a protein aggregation suppressor in the biopharmaceutical industry, its use has been questioned due to decreasing transition unfolding temperatures (Tm). Four compounds were designed to enhance the role of arginine by changing the length of the carbon chain with removal or N-acetylation of α-amino group. Biophysical properties were observed by differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow imaging (FI). N-Acetyl-L-arginine (NALA) performed the best at minimizing decrease in Tm with arginine at different pH. NALA also demonstrated relatively higher colloidal stability than arginine hydrochloride, especially in the acidic pH, thereby reducing agitation stress of IgG. Moreover, NALA exhibited a cooperative effect with commercially used glycine buffer for IVIG to maintain the monomer contents with almost no change and suppressed larger particle formation after agitation with heat. The study concludes that the decreasing Tm of proteins by arginine hydrochloride is due to amide group in the α-carbon chain. Moreover, chemical modification on the group compared to removing it will be a breakthrough of arginine's limitations and optimize storage stability of protein therapeutics.

Do not flick or drop off-label use plastic syringes in handling therapeutic proteins before administration.

This study investigates the formation of subvisible particles formed by external stresses produced such as flicking and dropping syringes. Flow imaging was used to visualize and quantify microparticles from 1 µm to over 25 µm as a result of mishandling. Microparticles increased in the presence of silicone oil that was present in syringes. Thus, silicone oil in syringes may affect the activity of therapeutic proteins being injected. Present data showed detailed and differentiated morphologies of proteinaceous particles, silicone oil, air bubbles, and plastic debris in mishandled syringes. In some cases, the presence of bisphenol A in syringes was detected by FT-IR. Disposable plastic syringes were evaluated and showed differences in their content of silicone oil. Syringes that contain 0.45 µm filters inside the needle cap as well as silicone oil-free syringes release proteinaceous subvisible particles after mechanical stress. These stress-generated particles can be delivered to patients, compromising patient care.

Therapeutic Efficacy of ABN401, a Highly Potent and Selective MET Inhibitor, Based on Diagnostic Biomarker Test in MET-Addicted Cancer.

The receptor tyrosine kinase c-MET regulates processes essential for tissue remodeling and mammalian development. The dysregulation of c-MET signaling plays a role in tumorigenesis. The aberrant activation of c-MET, such as that caused by gene amplification or mutations, is associated with many cancers. c-MET is therefore an attractive therapeutic target, and inhibitors are being tested in clinical trials. However, inappropriate patient selection criteria, such as low amplification or expression level cut-off values, have led to the failure of clinical trials. To include patients who respond to MET inhibitors, the selection criteria must include MET oncogenic addiction. Here, the efficacy of ABN401, a MET inhibitor, was investigated using histopathologic and genetic analyses in MET-addicted cancer cell lines and xenograft models. ABN401 was highly selective for 571 kinases, and it inhibited c-MET activity and its downstream signaling pathway. We performed pharmacokinetic profiling of ABN401 and defined the dose and treatment duration of ABN401 required to inhibit c-MET phosphorylation in xenograft models. The results show that the efficacy of ABN401 is associated with MET status and they highlight the importance of determining the cut-off values. The results suggest that clinical trials need to establish the characteristics of each sample and their correlations with the efficacy of MET inhibitors.