The stability of lyophilized lipid/DNA complexes during prolonged storage

It is well known that excipients are required to protect nonviral vectors during the lyophilization process. The goal of this study is to describe the stability of lyophilized nonviral vector preparations on pharmaceutically relevant timescales and provide insight into the factors that govern long-term stability of vectors in the dried state. Lipid/DNA complexes were lyophilized in glucose, sucrose, or trehalose and stored for a period of up to 2 years at five different temperatures (-20, 4, 22, 40, 60 degrees C). We evaluated simultaneously the physico-chemical characteristics (size, zeta potential, ethidium bromide (EtBr) accessibility, supercoiled DNA content) and the ability of vector formulations to transfect COS-7 cells at different time intervals. In addition, a fluorescence assay was utilized to assess levels of ROS in the dried cake after storage. The physical state of each formulation was evaluated by determination of the glass transition temperature and residual moisture content, before and after storage. Results from our stability study show that a progressive degradation of lipid/DNA complexes occurs in terms of transfection rates, particle size, dye accessibility, and supercoil content, even when samples are stored at low temperatures (e.g., -20 degrees C). Furthermore, our preliminary results on the quantification of free radicals in rehydrated formulations emphasize the importance of developing strategies to prevent the formation of reactive oxygen species (ROS) during prolonged storage in the dried state.     

Retrospective statistical analysis of lyophilized protein formulations of progenipoietin using PLS: determination of the critical parameters for long-term storage stability

Although certain criteria have become recognized as being essential for a stable lyophilized formulation, the relative importance of different stability criteria has not been demonstrated quantitatively. This study uses multivariate statistical methods to determine the relative importance of certain formulation variables that affect long-term storage stability of a therapeutic protein. Using the projection to latent structures (PLS) method, a retrospective analysis was conducted of 18 formulations of progenipoietin (ProGP), a potential protein therapeutic agent. The relative importance of composition, pH, maintenance of protein structure (as determined by infrared (IR) spectroscopy), and thermochemical properties of the glassy state (as measured by differential scanning calorimetry (DSC)) were evaluated. Various stability endpoints were assessed and validated models constructed for each using the PLS method. Retention of parent protein and the appearance of degradation products could be adequately modeled using PLS. The models demonstrate the importance of retention of native structure in the solid state and controlling the pH. The relative importance of T(g) in affecting storage stability was low, as all of the samples had T(g) values above the highest storage temperature (40 degrees C). However, other indicators of molecular mobility in the solid state, such as change in DeltaC(p) upon annealing, appear to be important, even for storage below T(g). For the first time, the relative importance of certain properties in controlling long-term storage stability could be assessed quantitatively. In general, the most important parameters appear to be pH and retention of native structure in the solid state. However, for some stability endpoints, the composition (concentration of protein or various excipients), as well as some DSC parameters, were found to be significant in predicting long-term stability.     

Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethyl starch mixtures

Optimal storage stability of a protein in a dry formulation depends on the storage temperature relative to the glass transition temperature (T(g)) of the dried formulation and the structure of the dried protein. We tested the hypothesis that optimizing both protein structure and T(g)--by freeze-drying recombinant human interleukin-11 (rhIL-11) with mixtures of disaccharides and hydroxyethyl starch (HES)--would result in increased storage stability compared with the protein lyophilized with either disaccharide or hydroxyethyl starch alone. The secondary structure of the protein in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures by infrared spectroscopy. After rehydration, aggregation was monitored by size exclusion chromatography. Oxidation levels and cleavage products were quantified by reversed-phase chromatography. For the formulation with HES alone, which has a relatively high T(g), storage stability of rhIL-11 was poor, because HES failed to inhibit lyophilization-induced unfolding. The sugar formulations inhibited unfolding, and had intermediate T(g) values and storage stabilities. Addition of hydroxyethyl starch to sucrose or trehalose increased T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization. Optimal storage stability of lyophilized rhIL-11 was achieved by using a mixture of disaccharide and polymeric carbohydrates.     

Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency

Cationic lipid-based gene delivery systems have shown promise in transfecting cells in vitro and in vivo. However, liposome/DNA complexes tend to form aggregates after preparation. Lyophilization of these systems, therefore, has become of increasing interest. In this study, we investigated the feasibility of preserving complexes as a dried preparation using a modified dehydration rehydration vesicle (DRV) method as a convenient and reliable procedure. We also studied storage stability of a lyophilized novel cationic gene delivery system incorporating sucrose, isomaltose and isomaltotriose. Liposomes were composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and L-dioleoylphosphatidylethanolamine (DOPE), plus sucrose, isomaltose or isomaltotriose. Lyophilized liposome/DNA complexes were stored at -20, 25, 40 and 50 degrees C and their stability was followed for 50 days. Liposome/DNA complexes with sucrose could be stored even at 50 degrees C without large loss of transfection efficiency. The transfection efficiency of formulations stored at various temperatures indicated that the stabilizing effect of sugars on plasmid DNA was higher in the following order: isomaltotriose相似文献   

Using dextran of different molecular weights to achieve faster freeze-drying and improved storage stability of lactate dehydrogenase

Freeze-drying of protein formulations is frequently used to maintain protein activity during storage. The freeze-drying process usually requires long primary drying times because the highest acceptable drying temperature to obtain acceptable products is dependent on the glass transition temperature of the maximally freeze-concentrated solution (T_g′). On the other hand, retaining protein activity during storage is related to the glass transition temperature (T_g) of the final freeze-dried product. In this study, dextrans with different molecular weight (1 and 40?kDa) and mixtures thereof at the ratio 3:1, 1:1, and 1:3 (w/w) were used as cryo-/lyoprotectant and their impact on the stability of the model protein lactate dehydrogenase (LDH) was investigated at elevated temperatures (40?°C and 60?°C). The dextran formulations were then compared to formulations containing sucrose as cryo-/lyoprotectant. Because of the higher T_g′ values of the dextrans, the primary drying times could be reduced compared to freeze-drying with sucrose. Similarly, the higher T_g and T_g′ of dextrans relative to sucrose led to benefits during storage which was shown through improved protection of LDH activity.     

A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.     

Long term stability of lyophilized plasmid DNA pDERMATT

In this short note we report on the shelf-life stability of pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) 2 mg lyophilized powder for reconstitution for intradermal administration, used in an in-house, investigator-initiated clinical phase I study. pDERMATT was stored at 25 °C/60% relative humidity (6 months), 2–8 °C (24 months), and −20 °C (66 months) in the dark and analyzed at several timepoints during the conduct of the clinical study for appearance, identity, purity (plasmid topology), content and residual water content. pDERMATT appeared stable at all storage conditions for the periods tested which, although patient inclusion in the study was significantly delayed, ensured the clinical supply needs. This study shows that lyophilization is an useful tool to preserve the quality of the pDNA and can prevent the need for costly and time-consuming additional manufacture of drug product in case of study delays, not uncommon at the early stage of drug development. To our knowledge, this is the first study reporting shelf life stability of a pDNA formulation for more than 5 years.     

Correlation of annealing with chemical stability in lyophilized pharmaceutical glasses

This research constitutes a thorough study of the relationship between the chemical stability, aging state and global molecular motion on the one hand, and microscopic local mobility in multi-component systems on the other hand. The objective of the present work was to determine whether annealing a glass below T(g) affects its chemical stability and determine if the rate of chemical degradation couples with global relaxation times determined using calorimetery, and/or with T(1) and T(1rho) relaxation times measured using ssNMR. Model compounds chosen for this research were lyophilized aspartame/sucrose and aspartame/trehalose (1:10 w/w) formulations. The chemical degradation was assessed at various temperatures using high-performance liquid chromatography (HPLC) to determine the impact of annealing on chemical stability. The rate constant for chemical degradation was estimated using stretched time kinetics. The results support the hypothesis that thermal history affects the molecular mobility required for structural relaxation and such effect is critical for chemical stability, that is, a stabilization effect upon annealing is observed.     

Structure, stability, and mobility of a lyophilized IgG1 monoclonal antibody as determined using second-derivative infrared spectroscopy

There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening β-relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular β-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the β-sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.     

Release stability of 5-fluorouracil liposomal concentrates, gels and lyophilized powder

Possible leakage of 5-fluorouracil from stable plurilamellar vesicles was monitored during storage of the liposomal concentrates, gels and lyophilized powders. Changes in release profile of dibucaine were taken as indicator of instability. Release profiles were obtained using the dialysis technique for a freshly prepared liposomal concentrate, gel or reconstituted lyophilized powder (zero time) and storage for one, two and four weeks in well closed tubes at 4 degrees C for the liposomal concentrate or gel and at 25 degrees C for liposomal lyophilized powder. Aiming at increasing stability of 5-fluorouracil liposomal dispersion, freshly prepared liposomal concentrates were directly incorporated in hydroxypropyl methylcellulose gel. Stability release profiles of liposomal gels and concentrates indicated a significant increase in stability of liposomal formulations. Also, lyophilization increases the shelf life of liposomes by preserving it in a dry form as a lyophilized cake to be reconstituted immediately prior to administration or direct incorporation into a final dosage form. Release and physico-chemical stability studies showed superior potentials of the lyophilized product after reconstitution in comparison to concentrate and gel forms. It could be concluded that lyophilization of liposomes loaded with a water-soluble drug such as 5-fluorouracil could significantly increase the stability of the liposomal vesicles and decrease leakage from it.     

Optimization of rapamycin-loaded acetalated dextran microparticles for immunosuppression

Immunosuppressive drugs can treat autoimmune disorders and limit rejection with organ transplants. However, delivering immunosuppressants like rapamycin systemically can have harmful side-effects. We aim to potentially reduce these toxic side-effects by encapsulating rapamycin in a polymeric microparticle to passively target phagocytes, the cells integral in immunosuppression. Acetalated dextran (Ac-DEX) is a recently described, biocompatible polymer which undergoes tunable burst degradation at the acidic conditions present in the phagosome (pH 5) but slower degradation at extracellular conditions (pH 7.4), thereby making it an ideal candidate for immune applications. Rapamycin-loaded microparticles were fabricated from Ac-DEX through a single emulsion (water/oil) technique. Optimized microparticles were determined by varying the chemical and physical parameters during particle synthesis. Microparticles synthesized from Ac-DEX with a molecular weight of 71 k had higher encapsulation efficiency of rapamycin and slower overall degradation than microparticles synthesized from 10k Ac-DEX. To evaluate the ability of rapamycin-loaded Ac-DEX microparticles to reduce a pro-inflammatory response, they were incubated with lipopolysaccharide-stimulated RAW macrophages. RAW macrophages treated with rapamycin-loaded microparticles exhibited reduced nitric oxide production and favorable cell viability. Overall, we have shown optimization of immunosuppressive rapamycin-loaded microparticles using the novel polymer Ac-DEX. These particles will be advantageous for future applications in immune suppression therapies.     

Short- and long-term stability study of lyophilized solid lipid nanoparticles for gene therapy

Most studies in gene therapy are focused on developing more efficient non-viral vectors, ignoring their stability, even though physically and chemically stable vectors are necessary to achieve large easily shipped and stored batches. In the present work, the effect of lyophilization on the morphological characteristics and transfection capacity of solid lipid nanoparticles (LyoSLN) and SLN-DNA vectors (Lyo(SLN-DNA)) has been evaluated. The lyophilized preparations were stored under three different sets of temperature and humidity ICH conditions: 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH. After lyophilization we found an increase in particle size which did not imply a reduction of “in vitro” transfection capacity. Stability studies of formulations lyophilized with trehalose showed that SLNs were physically stable during 9 months at 25 °C/60%RH and 6 months at 30 °C/65%RH. This stability was lost when harder conditions were employed (40 °C/75%RH). LyoSLNs maintained or increased the transfection efficacy (from 19% to approximately 40% EGFP positive cells) over time only at 25 °C/60%RH and 30 °C/65%RH. Lyo(SLN-DNA) resulted in almost no transfection under all conditions. LyoSLNs showed less DNA condensation capacity, whereas in Lyo(SLN-DNA) the plasmid became strongly bound, hampering the transfection. Furthermore, the storage of lyophilized lipoplexes stabilized with the disaccharide trehalose did not affect cell viability.     

Compatibility and stability of clindamycin phosphate-aminoglycoside combinations within polypropylene syringes

The stability and compatibility of clindamycin phosphate admixed separately with gentamicin sulfate, tobramycin sulfate, and amikacin sulfate in polypropylene syringes under specific storage conditions were studied. In duplicate syringes, clindamycin phosphate 900 mg was admixed with sterile NaCl 0.9% l ml and with either gentamicin sulfate 120 mg, tobramycin sulfate 120 mg, or amikacin sulfate 750 mg. In duplicate polypropylene syringes, control solutions of clindamycin phosphate and each aminoglycoside were prepared separately and stored under the same conditions. The clindamycin control consisted of clindamycin phosphate 900 mg in 6 ml. The gentamicin and tobramycin controls consisted of gentamicin sulfate and tobramycin sulfate 120 mg in 3 ml plus 1 ml of sterile NaCl 0.9%. The amikacin control consisted of amikacin sulfate 750 mg in 3 ml plus 1 ml of sterile NaCl 0.9%. Drug concentrations were determined at the time of preparation and 1, 4, 8, 12, 24, and 48 hours thereafter. Aminoglycosides were assayed by fluorescence polarization immunoassay and clindamycin was assayed by high performance liquid chromatography. Visual inspections and pH determinations of each combination and control solution were performed at each assay time. For the clindamycin, gentamicin, tobramycin, and amikacin control solutions, changes in concentration were within ten percent of the original concentration. Concentrations of clindamycin and gentamicin when admixed together also remained within ten percent of the original concentration. Similar results were found with concentrations of clindamycin and amikacin when admixed together. Tobramycin and clindamycin formed a lasting precipitate upon initial contact when admixed under the study conditions.     

Presence and stability of B complex vitamins in bee pollen using different storage conditions

This study has the objective of evaluating the stability of B complex vitamins and its vitamers, for a period of 1 year of storage. The pollen samples were stored under room temperature (with and without light) and frozen. The vitamins were quantified by HPLC with fluorescence detection. All proposed vitamins were found in the samples and the dehydration process did not interfere in vitamin content. The variations were (dry basis): 0.59–1.09 mg/100 g (B₁); 1.73–2.56 (B₂); 6.43–15.34 (PP) and 0.33–0.68 (B₆). After 1 year of storage, it can be stated that vitamin B₁ concentration remained constant, while for the others, the concentration loss was dependent on time rather than on storage conditions. All samples were considered vitamin B₂ sources. The influence of the storage time in the concentrations of vitamin B₆ and PP was explained mathematically, through linear regression equations of multivariate analysis.     

Optimization and evaluation of lyophilized fenofibrate nanoparticles with enhanced oral bioavailability and efficacy

Abstract

The objective of this study was to enhance physiochemical properties as well as oral bioavailability of the poorly water soluble drug fenofibrate (FB), through preparation of amorphous solid dispersions (ASDs). ASDs were prepared via freeze drying using polyvinylpyrrolidone (PVP) K30 and poloxamer 188 as hydrophilic carriers. Formulations were optimized by 3² full factorial design (FFD) with PVP-K30 level (X₁) and poloxamer 188 level (X₂) as independent variables and particle size (Y₁), zeta potential (Y₂), drug content (Y₃) and dissolution rate (T90, [Y₄]) as dependent variables. Optimized FB nanoparticles were physicochemically evaluated and formulated into lyophilized sublingual tablets. Pharmacokinetic, pharmacodynamics and histological finding of optimized formulation were performed on rabbits. Y₁ and Y₄ were significantly affected by independent variables while Y₂ and Y₃ were not affected. Physicochemical characterization showed the drug was in amorphous state, nanometer range and pharmacophore of FB was preserved. Administration of optimized FB tablets to rabbits with fatty liver led to significant reduction (p?<?0.001) in serum lipids. Moreover, histological analysis of liver specimens confirmed the improved efficacy in animals with fatty liver. In this study, we confirmed that ASDs of FB had beneficial effects on managing fatty liver and serum lipids level in hyperlipidemic rabbits.     

Preparation and storage stability of rutin nanosuspensions

Formulations of the rutin nanosuspensions have been prepared by high pressure homogenization (HPH). A Micron LAB 40 was used for HPH to obtain rutin nanosuspensions. Photon correlation spectroscopy (PCS) and laser diffraction were employed to analyze the particle size. Morphology of the particles was analyzed by light microscopy. The HPH technique produced rutin nanosuspensions having PCS size average of 547–912 nm and zeta potential range about ?30 mV in water. Aqueous rutin nanosuspension stabilized by SDS and Tween 80 were stable over 12 months. The nanosuspensions produced via HPH not only could prevent large particle size and particle growth, but also protect the drugs from chemically degradation. The molecules of the surface stabilizer are able to shield the chemical compound. The crystalline structure in a nanoparticulate sized formulation results in improved drug chemical-stability.     

Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples

Paralytic shellfish poison (PSP) has historically been a problem for the shellfish industry. In order to prevent the marketing of contaminated seafood products, governments have implemented monitoring programs where standards of toxins are necessary. The stability of these standard toxins is very important. In this paper we analysed the stability of saxitoxin (STX) and neosaxitoxin in acidic solution and lyophilized samples. Individual toxins were determined in each sample using a high-performance liquid chromatographic procedure employing post-column oxidation of the toxins to form fluorescent derivatives. Our results demonstrate that STX is very stable in solution samples and could be adopted as a reference standard. This toxin can be kept in dilute acidic solutions for 18 months without loss of potency. However, neosaxitoxin is unstable, possibly due to transformation to other toxins.     

Development and optimization of lyophilized orally disintegrating tablets using factorial design

The aim of this study was to evaluate the use of maltodextrin as a sugar-matrix former along with several cellulosic binders in the preparation of freeze—dried orally disintegrating tablets (ODT). The ODT was prepared by freeze—drying an aqueous dispersion of nimesulide (NM) containing maltodextrin and a cellulosic binder. The influence of formulation parameters on the in vitro/in vivo disintegration time and in vitro dissolution of NM from ODTs along with other tablet characteristics was investigated using full factorial design. The optimized ODT contained 5% w/v maltodextrin DE 29, 2% w/v Methocel^®E15, and 5% w/v NM, disintegrated in less than 10 s and showed more than 70% of NM in ODTs dissolved within 2?min, compared to only 1.52% of NM plain drug and 7.25% of NM in immediate release commercial tablet. Crystalline state evaluation of NM in the optimized ODT was conducted through differential scanning calorimetry, and X-ray powder diffraction. The study suggests that the optimized ODT formulation developed in this work may be an alternative to conventional formulations of NM inconvenient to the patients such as intramuscular or rectal administration.