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.

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.

Comparative study of lipid nanoparticle-based mRNA vaccine bioprocess with machine learning and combinatorial artificial neural network-design of experiment approach.

To develop a combinatorial artificial-neural-network design-of-experiment (ANN-DOE) model, the effect of ionizable lipid, an ionizable lipid-to-cholesterol ratio, N/P ratio, flow rate ratio (FRR), and total flow rate (TFR) on the outcome responses of mRNA-LNP vaccine were evaluated using a definitive screening design (DSD) and machine learning (ML) algorithms. Particle size (PS), PDI, zeta potential (ZP), and encapsulation efficiency (EE) of mRNA-LNP were optimized within a defined constraint (PS 40-100 nm, PDI ≤ 0.30, ZP≥(±)0.30 mV, EE ≥ 70 %), fed to ML algorithms (XGBoost, bootstrap forest, support vector machines, k-nearest neighbors, generalized regression-Lasso, ANN) and prediction was compared to ANN-DOE model. Increased FRR decreased the PS and increased ZP, while increased TFR increased PDI and ZP. Similarly, DOTAP and DOTMA produced higher ZP and EE. Particularly, a cationic ionizable lipid with an N/P ratio ≥ 6 provided a higher EE. ANN showed better predictive ability (R2 = 0.7269-0.9946), while XGBoost demonstrated better RASE (0.2833-2.9817). The ANN-DOE model outperformed both optimized ML models by R2 = 1.21 % and RASE = 43.51 % (PS prediction), R2 = 0.23 % and RASE = 3.47 % (PDI prediction), R2 = 5.73 % and RASE = 27.95 % (ZP prediction), and R2 = 0.87 % and RASE = 36.95 % (EE prediction), respectively, which demonstrated that ANN-DOE model was superior in predicting the bioprocess compared to independent models.

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.

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.

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.

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 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.

Basal buffer systems for a newly glycosylated recombinant human interferon-ß with biophysical stability and DoE approaches.

The purpose of this study was to develop a basal buffer system for a biobetter version of recombinant human interferon-ß 1a (rhIFN-ß 1a), termed R27T, to optimize its biophysical stability. The protein was pre-screened in solution as a function of pH (2-11) using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the result, its experimental pI and optimal pH range were 5.8 and 3.6-4.4, respectively. Design of experiment (DoE) approach was developed as a practical tool to aid formulation studies as a function of pH (2.9-5.7), buffer (phosphate, acetate, citrate, and histidine), and buffer concentration (20 mM and 50 mM). This method employed a weight-based procedure to interpret complex data sets and to investigate critical key factors representing protein stability. The factors used were Tm, enthalpy, and relative helix contents which were obtained by DSC and Fourier Transform Infrared spectroscopy (FT-IR). Although the weights changed by three responses, objective functions from a set of experimental designs based on four buffers were highest in 20 mM acetate buffer at pH 3.6 among all 19 scenarios tested. Size exclusion chromatography (SEC) was adopted to investigate accelerated storage stability in order to optimize the pH value with susceptible stability since the low pH was not patient-compliant. Interestingly, relative helix contents and storage stability (monomer remaining) increased with pH and was the highest at pH 4.0. On the other hand, relative helix contents and thermodynamic stability decreased at pH 4.2 and 4.4, suggesting protein aggregation issues. Therefore, the optimized basal buffer system for the novel biobetter was proposed to be 20 mM acetate buffer at pH 3.8±0.2.

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.