Protein Storage Stability Research Articles

Lanmodulin-Functionalized Magnetic Nanoparticles as a Highly Selective Biosorbent for Recovery of Rare Earth Elements.

Recovering rare earth elements (REEs) from waste streams represents a sustainable approach to diversify REE supply while alleviating the environmental burden. However, it remains a critical challenge to selectively separate and concentrate REEs from low-grade waste streams. In this study, we developed a new type of biosorbent by immobilizing Lanmodulin-SpyCatcher (LanM-Spycatcher) on the surface of SpyTag-functionalized magnetic nanoparticles (MNPs) for selective separation and recovery of REEs from waste streams. The biosorbent, referred to as MNP-LanM, had an adsorption activity of 6.01 ± 0.11 μmol-terbium/g-sorbent and fast adsorption kinetics. The adsorbed REEs could be desorbed with >90% efficiency. The MNP-LanM selectively adsorbed REEs in the presence of a broad range of non-REEs. The protein storage stability of the MNP-LanM increased by two-fold compared to free LanM-SpyCatcher. The MNP-LanM could be efficiently separated using a magnet and reused with high stability as it retained ∼95% of the initial activity after eight adsorption-desorption cycles. Furthermore, the MNP-LanM selectively adsorbed and concentrated REEs from the leachate of coal fly ash and geothermal brine, resulting in 967-fold increase of REE purity. This study provides a scientific basis for developing innovative biosorptive materials for selective and efficient separation and recovery of REEs from low-grade feedstocks.

Storage stability of proteins in a liquid-based formulation: Liquid vs. solid self-emulsifying drug delivery.

The objective of the present study was the development of self-emulsifying drug delivery systems (SEDDS) for oral delivery of therapeutic proteins providing storage stability. Horseradish peroxidase (HRP) serving as model protein was ion paired with docusate and incorporated into three liquid and three solid SEDDS formulations. Storage stability of HRP was determined over three weeks by quantifying its enzymatic activity. Generally, HRP maintained 78% of its initial enzymatic activity after complexation and loading into SEDDS. Having been incorporated in liquid SEDDS the protein showed limited stability and precipitated within a few hours. In contrast, in all solid SEDDS comprising of hard fats such as Witepsol W45 and solid surfactants such as Gelucire 44/14 and 48/16 as solidifying agents HRP was successfully stabilized. No decrease in HRP activity could be observed over the entire observation period. Solid SEDDS based on high-melting components can provide storage stability of incorporated proteins, whereas liquid SEDDS cannot.

Formulating monoclonal antibodies as powders for reconstitution at high concentration using spray-drying: Trehalose/amino acid combinations as reconstitution time reducing and stability improving formulations

To increase their stability, therapeutic (or monoclonal) antibodies (mAbs) are often formulated as solids by using a variety of drying techniques, e.g. freeze-drying, spray-drying, or spray freeze-drying. The addition of excipients is required to preserve stability of the protein during the drying process and subsequent storage of the resulting solid form. The addition of low molecular weight excipients, such as amino acids, to sugar based spray- and freeze-dried formulations has been suggested to improve the storage stability of proteins in the dried state. In this study sugars (sucrose, trehalose), amino acids (Gly, Ala, Pro, Ser, Val, Leu, Ile, Gln, His, Lys, Arg, Phe, Trp) and combinations thereof were investigated for their stabilizing effect during spray-drying and subsequent storage and for their reconstitution time reducing effect. Two IgG4 mAbs were used as model antibodies.From an initial screening study, basic and small neutral amino acids, in combination with a sugar, such as sucrose or trehalose, showed reconstitution time reducing and stabilizing properties. Arg in particular displayed excellent reconstitution and stability enhancing properties. Moreover, Arg was the only amino acid providing stabilizing properties comparable to sucrose or trehalose. Previous work by the authors described a statistically substantiated comparison between the three basic amino acids in a sugar containing formulation, albeit limited to a single concentration level [5]. Therefore, a follow-up design of experiments (DoE) study was performed to determine the optimum trehalose/amino acid content required for an optimal protein stability and reconstitution time and to compare the effects of two basic amino acids, Lys and Arg, to those of two neutral amino acids, Gly and Pro. The conducted DoE covered a wide range of trehalose (30–120 mM) and amino acid (50–150 mM) concentrations. The concentration of trehalose was found to be the main contributor to a reduction in reconstitution time and an increase in stability. Here we show that the addition of amino acids such as Gly, Pro, and Lys does not improve stability nor does it reduce the reconstitution time. Of the tested amino acids, only Arg showed a marked reduction in reconstitution time and improvement in stability compared to a trehalose. Moreover, the properties displayed by Arg could justify its application as the main stabilizer in spray-dried mAb formulations, eliminating the need for a sugar matrix altogether. But the weight ratio of stabilizer to protein was found the factor exerting the strongest overall influence on the formulation’s reconstitution time and stability. More specifically, sufficient physical stability and an acceptable reconstitution time could be obtained with a protein to stabilizer weight ratio of at least 1:1.

Binding of excipients is a poor predictor for aggregation kinetics of biopharmaceutical proteins

One of the major challenges in formulation development of biopharmaceuticals is improving long-term storage stability, which is often achieved by addition of excipients to the final formulation. Finding the optimal excipient for a given protein is usually done using a trial-and-error approach, due to the lack of general understanding of how excipients work for a particular protein. Previously, preferential interactions (binding or exclusion) of excipients with proteins were postulated as a mechanism explaining diversity in the stabilisation effects. Weak preferential binding is however difficult to quantify experimentally, and the question remains whether the formulation process should seek excipients which preferentially bind with proteins, or not. Here, we apply solution NMR spectroscopy to comprehensively evaluate protein-excipient interactions between therapeutically relevant proteins and commonly used excipients. Additionally, we evaluate the effect of excipients on thermal and colloidal protein stability, on aggregation kinetics and protein storage stability at elevated temperatures. We show that there is a weak negative correlation between the strength of protein-excipient interactions and effect on enhancing protein thermal stability. We found that the overall protein-excipient binding per se can be a poor criterion for choosing excipients enhancing formulation stability. Experiments on a diverse set of excipients and test proteins reveal that while excipients affect all of the different aspects of protein stability, the effects are very much protein specific, and care must be taken to avoid apparent generalisations if a smaller dataset is being used.

Improvement in bioactive protein storage stability and colon-targeted release: a simple double-layer chitosan-based particle

Aim: To improve the long-term storage stability of a bioactive protein and the subsequent colon-targeted release, a double-layer chitosan (CS)-based particle was developed.Methods: To form the double-layer CS-based particle, the second layer was crosslinked onto the single-layer bovine serum albumin (BSA)-loaded CS-based particle. The structure and properties of particles were further investigated.Results: With the second layer, the double-layer particles became more compact, which was important for the inhibition of bioactive protein leakage during storage through strong electrostatic interactions and swelling of the hydrogels. After 30 d of storage, there was only 43.74–49.32% BSA leakage from the C15-TPP/C15-HMP double-layer particles. Moreover, the BSA release in subsequent colon-targeted delivery after storage was 44.02–48.59%.Conclusions: With double-layer shielding and a more compact arrangement, it was possible to reduce bioactive protein leakage over long periods storage and achieve subsequent colon-targeted delivery.

Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations.

Freeze-drying is a popular method to prepare pharmaceutical formulations containing structurally complex active ingredients and drug delivery system carriers. The solidification performed at lower temperature significantly improves storage stability of proteins, peptides, antibiotics, vaccines, and liposomes, which are marginally stable in aqueous solutions. Individual components of the freeze-drying process (freezing, primary drying, secondary drying), however, expose proteins to various stresses. Certain excipients, including disaccharides (e.g., sucrose, trehalose) and amino acids, can be added to protect the proteins and supramolecular drug delivery systems against physical stress associated with freezing and storage by substituting the molecular interactions provided by water molecules. Some excipients embed the active ingredients in glass-state solids with low molecular mobility, thereby reducing chemical reactivity. Thus, the use of appropriate excipients and process control is important to protect proteins during freeze-drying. This chapter describes the applications of freeze-drying in the pharmaceutical production process, mainly focusing on formulation and process optimization for protein therapeutics.

Mannitol/l-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals: Effect of the l-Arginine Counter Ion and Formulation Composition on the Formulation Properties and the Physical State of Mannitol

Previous studies have shown that protein storage stability in freeze-dried l-arginine-based systems improved in the presence of chloride ions. However, chloride ions reduced the glass transition temperature of the freeze concentrate (Tg′) and made freeze drying more challenging. In this study, l-arginine was freeze dried with mannitol to obtain partially crystalline solids that can be freeze dried in a fast process and result in elegant cakes. We characterized the effect of different l-arginine counter ions on physicochemical properties of mannitol compared with mannitol/sucrose systems. Thermal properties of formulations with different compositions were correlated to thermal history during freeze drying and to physicochemical properties (cake appearance, residual moisture, reconstitution time, crystallinity). Partially crystalline solids were obtained even at the highest l-arginine level (mannitol:l-arginine of 2:1) used in this study. All l-arginine-containing formulations yielded elegant cakes. Only cakes containing l-arginine chloride and succinate showed a surface “crust” formed by phase separation. X-ray powder diffraction showed that inhibition of mannitol crystallization was stronger for l-arginine compared with sucrose and varied with the type of l-arginine counter ion. The counter ion affected mannitol polymorphism and higher levels of mannitol hemi-hydrate were obtained at high levels of l-arginine chloride.

Influence of Miscibility of Protein-Sugar Lyophilizates on Their Storage Stability.

For sugars to act as successful stabilizers of proteins during lyophilization and subsequent storage, they need to have several characteristics. One of them is that they need to be able to form interactions with the protein and for that miscibility is essential. To evaluate the influence of protein-sugar miscibility on protein storage stability, model protein IgG was lyophilized in the presence of various sugars of different molecular weight. By comparing solid-state nuclear magnetic resonance spectroscopy relaxation times of both protein and sugar on two different timescales, i.e., (1)H T1 and (1)H T1ρ, miscibility of the two components was established on a 2-5- and a 20-50-nm length scale, respectively, and related to protein storage stability. Smaller sugars showed better miscibility with IgG, and the tendency of IgG to aggregate during storage was lower for smaller sugars. The largest sugar performed worst and was phase separated on both length scales. Additionally, shorter protein (1)H T1 relaxation times correlated with higher aggregation rates during storage. The enzyme-linked immunosorbent assay (ELISA) assay showed overlapping effects of aggregation and chemical degradation and did not correspond as well with the miscibility. Because of the small scale at which miscibility was determined (2-5nm) and the size of the protein domains (∼2.5 × 2.5 × 5nm), the miscibility data give an indirect measure of interaction between protein and sugar. This reduced interaction could be the result of steric hindrance, providing a possible explanation as to why smaller sugars show better miscibility and storage stability with the protein.

In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formation between protein and sugar, predictive of protein storage stability

Sugars are often used as stabilizers of protein formulations during freeze-drying. However, not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy during freeze-drying, it is shown here that hydrogen bond formation during freeze-drying, under secondary drying conditions in particular, can be related to the preservation of the functionality and structure of proteins during storage. The disaccharide trehalose was best capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4kDa. The molecularly rigid oligo- and polysaccharides dextran 5kDa and 70kDa, respectively, formed the least amount of hydrogen bonds and provided least stabilization of the protein. It is concluded that smaller and molecularly more flexible sugars are less affected by steric hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it better.

DESIGN OF COMPOSITION OF ACTIVE PHARMACEUTICAL INGREDIENTS BASED ON E7-HSP70 HYBRID RECOMBINANT PROTEINS

The approaches to the stabilization of the recombinant fusion oncoproteins E7 16 and 18 conjugated with heat shock protein70 insolutions have been studied. The screening of pharmaceutical active substances was carried out by the accelerated storage test at elevated temperatures. The first stage of the study was aimed at determining of the pH range providing the best stability of the physicochemical quality parameters of the proteins which turned to be 6.8—7.4. The second and third steps were aimed at identification of excipients that make the greatest contribution to the increased storage stability of proteins in solution showed that sucrose and polysorbate 80 constitute the most stable composition. The temperature-20 °Callows to keep the quality parameters on a desired level for at least 12 months.

Impact of sucrose level on storage stability of proteins in freeze-dried solids: I. correlation of protein–sugar interaction with native structure preservation

The purpose of this study is to investigate protein–sugar interactions in dried protein solids as a function of sucrose level using water sorption isotherm data and secondary structure information from Fourier transform infrared (FTIR) spectroscopy. Three IgG1 fusion proteins and two cytokines were freeze-dried with sucrose at different sucrose/protein mass ratios. The water monolayer of the colyophilized sucrose/protein samples, as determined by BET analysis of water sorption data, was found to be lower than that expected based on additive contributions of pure protein and pure sucrose. This negative deviation suggests the presence of a solid-state interaction between protein and sucrose that reduces the availability of total water-binding sites. The difference in water monolayer between colyophilized and a physical mixture of protein and sucrose reached a maximum value at sucrose/protein mass ratio of 1/1 for these proteins, suggesting saturation of the protein–sugar interaction at this ratio. In addition, for four proteins studied, the normalized peak height of the major band in the FTIR spectra reached a plateau at about a 1/1 mass ratio. Therefore, it appears that there is a coupling between the preservation of protein secondary structure and the protein–sugar interaction as measured by water sorption isotherms. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3131–3144, 2009

Optimization of Storage Stability of Lyophilized Actin Using Combinations of Disaccharides and Dextran

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting Tg of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and Tg by freeze‐drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in Tg observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation Tg without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 199–214, 2000

Stability and surface activity of lactate dehydrogenase in spray‐dried trehalose

The stability of the model protein lactate dehydrogenase (LDH) during spray‐drying and also on subsequent dry storage was examined. Trehalose was used as a carrier. The spray‐drying temperatures Tinlet and Toutlet have a measurable effect on LDH inactivation. Low Tinlet produced the least process inactivation, but gave a high residual moisture content making the protein's storage stability poor. High Tinlet reduced residual moisture and improved storage stability, but at the cost of high process inactivation. As already found for other systems, addition of a surfactant (in this case polysorbate 80) could ameliorate process inactivation of LDH at Tinlet = 150 °C. Surfactant had, however, a deleterious effect on storage stability of LDH, the vital factor being the molar ratio of surfactant/protein in the dried product. By using electron spectroscopy it was shown that LDH has a 10 times higher surface concentration in the dried trehalose particles than expected for a homogeneous distribution. Surface tension measurements at the water/air interface proved that LDH is surface active, although the Gibbs equation appeared to be inapplicable. Calculations of spray‐droplet formation time and drying time indicate than the extent of diffusion‐driven LDH adsorption to the liquid/air interface is sufficient to account for the measured amount of LDH inactivation during spray‐drying. The presence of 0.1% polysorbate 80 to the spray solution prevents LDH from appearing at the surface of the dried particles. As a negative control, the phosphatide Lipoid E 80 does not prevent the appearance of LDH in the surface according to electron spectroscopy and does not therefore prevent LDH inactivation during spray‐drying at Tinlet = 150 °C.

Physical Factors Affecting the Storage Stability of Freeze-Dried Interleukin-1 Receptor Antagonist: Glass Transition and Protein Conformation

The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56°C, during several weeks of storage at temperatures above and below the samples’ Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 ± 2.5°C. With infrared spectroscopy, we determined that the protein lyophilized with ≤1% sucrose was unfolded in the initial dried solid. In contrast, in those formulations with ≥5% sucrose, conformational change was inhibited during lyophilization. When stored at 50°C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation.