Comparison of product drying performance in molded and serum tubing vials using gentamicin sulfate as a model system

In a previous study, heat transfer coefficients of different 10?mL tubing and molded vials were determined gravimetrically via sublimation tests with pure water. Contrary to “conventional wisdom”, only small differences in K_v values between tubing and molded vials were found in the pressure range relevant for pharmaceutical freeze-drying. In order to investigate the impact of these relatively small differences on the primary drying time of an actual product, freeze-drying experiments with 5% gentamicin sulfate solution as a model system were performed at 68, 100 and 200 mTorr. The primary drying times of the API in recently developed molded (EasyLyo?), tubing (TopLyo?) and polymer vials (TopPac?) were compared. At 68 and 100 mTorr the primary drying time of the drug in the glass vials only differed by 3% to 4%, while the polymer vial took around 9% longer. At 200 mTorr, the API in the EasyLyo? vials dried approximately 15% faster compared to the other vial types. The present study suggest that molded vials that have been modified in design to have better heat transfer properties can achieve drying times comparable to tubing vials.     

The need for new control strategies for particulate matter in parenterals

An undesirable characteristic in lyophilized parenteral products is the potential presence of particulate matter in the final product, which may affect patient safety. In this study, quality risk management tools described in the International Conference on Harmonization Guideline Q9 were used to estimate the risks for a pharmaceutical manufacturing line, based on three critical quality attributes: (1) visible particulate matter; (2) lyo-cake collapse traces; and (3) lyo-cake melt-back traces. Together with a Process Failure Mode Effect Analysis (PFMEA), an input-output analysis of the individual unit operations identified seven major material classes of extrinsic particulate matter. In addition to the process assessment, an experimental investigation of the location of impurities in lyophilized products was performed. To that end, intentionally contaminated vials were examined to locate the particulate matter and its possible migration. The results emphasize the importance of a full transmission mode release testing since the particles may enter the interior of the lyo-cake. A theoretical explanation of the observed impurity locations is provided.     

Effects of Excipients on Protein Conformation in Lyophilized Solids by Hydrogen/Deuterium Exchange Mass Spectrometry

Purpose Excipients are added to lyophilized protein drug formulations to protect the protein during processing and storage, but the mechanisms are poorly understood. Here, hydrogen/deuterium (H/D) exchange with mass spectrometry was used to assess protein conformation and excipient interactions in lyophilized solids. Methods Calmodulin (CaM, 17 kD) was co-lyophilized with carbohydrate excipients (sucrose, mannitol, trehalose, raffinose, dextran 5,000, dextran 12,000) or guanidine hydrochloride (negative control) and exposed to D₂O vapor at 33% RH and RT. Samples were then dissolved and analyzed by mass spectrometry (+ESI/MS). Peptic digestion provided additional, site-specific information on H/D exchange. Solids were further characterized by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and water vapor sorption. Results Excipients protected CaM from H/D exchange, increasing in the order guanidine hydrochloride < no excipient, mannitol < dextran 5,000, dextran 12,000 < sucrose < raffinose < trehalose. Effects were exerted primarily in the protein’s α-helical segments. Conclusions The effects of carbohydrate excipients on protein conformation in lyophilized solids are not exhibited uniformly along the protein sequence, but instead are exerted in a site-specific manner. The results also demonstrate the utility of H/D exchange with ESI/MS for protein structure characterization in lyophilized samples.     

Poly (ethylene glycol) hydrogel scaffolds with multiscale porosity for culture of human adipose-derived stem cells

Three-dimensional (3?D) hydrogel scaffolds are an attractive option for tissue regeneration applications because they allow for cell migration, fluid exchange, and can be synthesized to closely mimic the physical properties of the extracellular matrix environment. The material properties of hydrogels play a vital role in cellular migration and differentiation. In light of this, in-depth understanding of material properties is required before such scaffolds can be used to study their influence on cells. Herein, various blends and thicknesses of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogels were synthesized, flash frozen, and dried by lyophilization to create scaffolds with multiscale porosity. Environmental scanning electron microscopy (ESEM) images demonstrated that lyophilization induced microporous voids in the PEGDMA hydrogels while swelling studies show the hydrogels retain their innate swelling properties. Change in pore size was observed between drying methods, polymer blend, and thickness when imaged in the hydrated state. Human adipose-derived stem cells (hASCs) were seeded on lyophilized and non-lyophilized hydrogels to determine if the scaffolds would support cell attachment and proliferation of a clinically relevant cell type. Cell attachment and morphology of the hASCs were evaluated using fluorescence imaging. Qualitative observations in cell attachment and morphology of hASCs on the surface of the different hydrogel spatial configurations indicate these multiscale porosity hydrogels create a suitable scaffold for hASC culture. These findings offer another factor of tunability in creating biomimetic hydrogels for various tissue engineering applications including tissue repair, regeneration, wound healing, and controlled release of growth factors.     

BIOPHARMACEUTICAL EVALUATION OF THE LIPOSOMES PREPARED BY REHYDRATION OF FREEZE-DRIED EMPTY LIPOSOMES (FDELs) WITH AN AQUEOUS SOLUTION OF A DRUG

We have evaluated a method for preparation of a dispersion of liposomes encapsulating a drug, namely rehydration of freeze-dried empty (not containing drug) liposomes with an aqueous drug solution (FDEL method). In the present study, we characterized and compared this method with the conventional method using a lipid composition of DPPC–DPPG–cholesterol in a molar ratio of 27:3:20. Two hydrophilic compounds, [³H]-inulin and [³H]-mannitol, were used as model drugs. Liposomal preparations by the FDEL method had an encapsulation efficiency of 2.9 and 6.7% for [³H]-inulin and [³H]-mannitol, respectively, when rehydrated and incubated at 70 °C. Since non-specific adsorption of these markers to liposomal membrane is negligible, this method produces liposomes which encapsulate a drug in the intravesicular space. One-tenth of the marker encapsulated in the liposomes prepared by the FDEL method (F-liposomes) was released very rapidly on incubation with rat plasma, followed by the slow release of the remaining fraction thereafter. No such rapid-release phase was observed for the liposomes prepared by the conventional method (C-liposomes). This suggests the existence of two types of encapsulation, loose encapsulation and tight encapsulation, in F-liposomes at least. Pharmacokinetic parameters of marker encapsulated tightly in F-liposomes were comparable to those in C-liposomes. It is likely that amphipathic drugs such as doxorubicin are incorporated into liposomes more easily than inulin and mannitol when formulated by the FDEL method. These results therefore suggest that the FDEL method is useful in the preparation of a liposomal formulation of a drug.     

Lyophilization provides long-term stability for a lipid nanoparticle-formulated,nucleoside-modified mRNA vaccine

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Production of a thermostable vero cell-adapted rinderpest vaccine

Summary The method of vaccine virus production, chemical stabilization, and lyophilization of a thermostable Vero cell-adapted rinderpest vaccine is presented. The production method results in a vaccine which can be used in the field without refrigeration for up to 30 d. The work was supported in part by grant 683-0242 from the U.S. Agency for International Development to the Government of the Republic of Niger.     

The Effects of Formulation Additives on the Degradation of Freeze-Dried Ribonuclease A

The stability of a freeze-dried model protein, ribonuclease A (RNase), was investigated under accelerated storage conditions at 45°C for time periods up to 60 days. Because RNase is a fairly stable molecule around pH 7, lyophilization was performed in phosphate buffers at pH 4.0 or 10.0 to accelerate degradation kinetics. Degradation was studied by measuring enzymatic activity, the concentrations of soluble monomeric RNase, soluble aggregated (polymerized) RNase, and insoluble aggregated RNase following reconstitution of the lyophilized material at different times. The presence of air in the vial headspace accelerated degradation in the solid state in all cases. When argon or nitrogen was employed in the headspace, degradation kinetics were reduced, implying that molecular oxygen was involved in the degradation process. This interpretation was supported by the observation that 0.05% (w/v) EDTA in the formulation prior to freeze-drying retarded RNase degradation dramatically. EDTA was believed to chelate cations which may have been introduced with the buffer salts in trace quantities sufficient to catalyze autoxidation reactions. Incorporation of antioxidants ascorbic acid (at pH 4.0) and POBN (a spin trap which could have functioned as an antioxidant at pH 10.0) accelerated the degradation of RNase and appeared, in both cases, to be involved in interactions with the protein molecules. Additionally, in the presence of the antioxidants RNase degradation appeared to be accelerated by light. Although there is strong support for the oxidative hypothesis, the possibility of other competing reactions cannot be discounted. These investigations demonstrate the importance of challenging the extrapolation of some of our well-established ideas concerning small molecule solution kinetics to macromolecules in the solid state.     

一种新型脂质体的制作方法

目的制备独立于药物的新型脂质体。方法在脂质体悬液中加入骨架物,冻干,制成超微孔分子膜,遇水即可成 脂质体。结果和结论　冻干法制备的脂质体便于运输、贮存,药物包裹简易。     

Noncontact Infrared-Mediated Heat Transfer During Continuous Freeze-Drying of Unit Doses

Recently, an innovative continuous freeze-drying concept for unit doses was proposed, based on spinning the vials during freezing. An efficient heat transfer during drying is essential to continuously process these spin frozen vials. Therefore, the applicability of noncontact infrared (IR) radiation was examined. The impact of several process and formulation variables on the mass of sublimed ice after 15 min of primary drying (i.e., sublimation rate) and the total drying time was examined. Two experimental designs were performed in which electrical power to the IR heaters, distance between the IR heaters and the spin frozen vial, chamber pressure, product layer thickness, and 5 model formulations were included as factors. A near-infrared spectroscopy method was developed to determine the end point of primary and secondary drying. The sublimation rate was mainly influenced by the electrical power to the IR heaters and the distance between the IR heaters and the vial. The layer thickness had the largest effect on total drying time. The chamber pressure and the 5 model formulations had no significant impact on sublimation rate and total drying time, respectively. This study shows that IR radiation is suitable to provide the energy during the continuous processing of spin frozen vials.