红细胞在保护剂添加和洗涤时的非平衡渗透响应实验研究

在红细胞低温保存或冻干保存中，高浓度保护剂的添加和洗涤会使细胞体积收缩或膨胀，造成溶血损失，但其体积并非单调收缩或膨胀到最后平衡体积，而是要经历更严重的体积变化后再趋于平衡。本研究以NaCl溶液来模拟保护剂的添加与洗涤过程．将38种不同浓度的NaC;溶液，以一步直接法、4步等体积法或4步等摩尔浓度变化法添加到红细胞中，测定其溶血率。若以溶血率10％为标准，红细胞所能承受的最小渗透压在161mOsmol／k附近，而最大渗透压与溶液添加方式有关，等体积添加法效果较好（其值约为5680mOsmol／kg）。研究中还将12％、18％NaCl溶液以4步等体积法加到红细胞中，再用生理盐水以三种方式洗涤，发现等摩尔浓度变化法洗涤效果最好；洗涤后溶血率大大增加，说明很多细胞虽然在保护剂添加时未溶血，但膜脆性已改变，难以恢复到等渗时体积。     

Effects of Product Handling Parameters on Particle Levels in a Commercial Factor VIII Product: Impacts and Mitigation

To reduce the risk of immunogenicity that may be caused by therapeutic protein products, it is important to properly characterize subvisible particles and to develop strategies to reduce the levels of particles delivered to patients. In the present study, by using state-of-the-art methods to quantify particle levels, we found that the factor VIII product, Kogenate FS, contained relatively high levels of protein particles and silicone oil droplets, the vast majority of which were submicron in size. In a test of effects of product mishandling, the Kogenate FS vial was shaken instead of swirled during reconstitution. Levels of silicone oil droplets and protein particles were increased. In contrast, these levels were greatly reduced by 2 mitigation strategies tested, using a nonsiliconized syringe for the diluent container or using submicron pore size syringe filters during simulated infusion. Thus, to avoid potential adverse effects due to mishandling-induced increases in particle levels, it is important to educate end-users about proper product handling. Furthermore, effective particle mitigation and reduction strategies should be developed for factor VIII, and other therapeutic protein products. Such efforts could lead to clinically useful approaches to reduce the levels of particles delivered to patients and to an associated reduction in adverse immunogenicity.     

Determining Maximum Sublimation Rate for a Production Lyophilizer: Computational Modeling and Comparison With Ice Slab Tests

Equipment capability is an important factor in scale up and technology transfer for lyophilized pharmaceutical products. Experimental determination of equipment capability limits, such as the maximum sublimation rate at a given chamber pressure, is time-intensive for production lyophilizers. Here, we present computational fluid dynamics modeling of equipment capability and compare it with experimental data for minimum controllable pressure ice slab sublimation tests in a 23 m² shelf area freeze dryer. It is found that the vapor flow in the production scale is characterized by turbulent effects at high sublimation rates. For the considered freeze dryer configuration, the onset of turbulence occurs at a sublimation rate of 17 kg/h and leads to an increase in the minimum controllable pressure by 3-4 mTorr for the flow rates up to 40 kg/h. Variations in the shelf and duct orientations as well as the valve stroke distance and their effect on the equipment limit and pressure uniformity are also discussed. The minimum controllable pressure measured experimentally agreed within 5% with computational fluid dynamics results. For high vapor sublimation rates at final stages of ice slab testing, the condenser load affects the product chamber pressure control. Estimate of condenser pressure changes because of ice accumulation has been included.     

Lyophilization decreases the formation of dialyzable iron by extraction and digestion of chicken breast muscle

We studied the effect of lyophilization of chicken breast muscle on the formation of dialyzable iron from ferric iron. Chicken breast muscle was used chilled, frozen or lyophilized and was analyzed for sulfhydryl and histidine content. It was then homogenized and mixed with ferric iron. The mixture was extracted with acid or digested with pepsin and pancreatin. The extracts and digests were analyzed for dialyzable ferrous and dialyzable total iron and also for protein. In the chilled muscle, similar amounts of dialyzable iron were formed after acid extraction and after proteolytic digestion; however, digestion led to more dialyzable ferrous iron. Freezing had no effect but lyophilization of the homogenized muscle caused large decreases in dialyzable iron and dialyzable ferrous iron for both extraction and digestion processes. Lyophilization also resulted in decreased extraction of peptides, decreased digestion of muscle proteins and reduced levels of sulfhydryl and histidine residues. Our results demonstrate that dialyzable iron is produced both by acid-soluble low molecular weight muscle component(s) and also by peptides resulting from digestion of muscle proteins: both of which reduce and chelate iron. Reduced formation of dialyzable iron by both mechanisms following lyophilization could be explained by sulfhydryl oxidation and impaired digestion due to protein crosslinking.     

生物膜的保护剂海藻糖在冻干红细胞中的应用

海藻糖作为一种稳定的非还原性双糖，在细胞冷冻、干燥、冻干过程中对细胞活性有着良好的保护作用并已广泛应用于人血细胞的冻干．海藻糖以其异常的水合能力、独特的玻璃化转换和晶体转变行为及抗高温潮湿的特性受到密切关注，并成为细胞保存研究中首选的一种保护剂。海藻糖对细胞的保护机制、海藻糖导入哺乳细胞的实验方法以及红细胞对海藻糖负载的可能作用机制部是近几年细胞保存研究的热点，本文就以上几方面的进展详细进行论述.     

Kinetics and Mechanism for the Reaction of Cysteine with Hydrogen Peroxide in Amorphous Polyvinylpyrrolidone Lyophiles

Purpose Peroxide impurities play a critical role in drug oxidation. In metal-free aqueous solutions, hydrogen peroxide (H₂O₂) induced thiol oxidation involves a bimolecular nucleophilic reaction to form a reactive sulfenic acid intermediate (RSOH), which reacts with a second thiol to form a disulfide (RSSR). This study examines the reaction of cysteine (CSH) and H₂O₂ in amorphous polyvinylpyrrolidone (PVP) lyophiles to explore the possible relevance of the solution mechanism to reactivity in an amorphous glass.Materials and Methods Amorphous PVP lyophiles containing CSH and H₂O₂ at varying initial ‘pH’ and reactant concentrations were prepared by methods designed to minimize reaction during lyophilization. Kinetic studies were conducted anaerobically at 25°C and reactants and products were monitored by HPLC. Products were characterized and the kinetic data were fit to models adapted from the solution mechanism.Results Key differences in the reactions in aqueous solution and amorphous PVP are: (1) while only cystine (CSSC) forms in solution, three degradants—cysteine sulfinic acid (CSO₂H), cysteine sulfonic acid (CSO₃H) and cystine (CSSC)—form in amorphous PVP; (2) simple bimolecular kinetics govern the solution reaction while initial rates in amorphous PVP suggested more complex kinetics (i.e., non-unity values for reaction order); and (3) heterogeneous (i.e., biphasic) reaction dynamics are evident in amorphous PVP. The differences in product formation and apparent reaction orders in the solid-state could be rationalized by partitioning of the same reactive intermediate to multiple products in the solid-state due to the restricted mobility of CSH. Beyond the initial rate region, the kinetics in amorphous PVP could be described by the Kohlrausch‐Williams‐Watts (KWW) stretched-exponential equation or by assuming two populations of reactant molecules having different reactivities.Conclusions When reactive intermediates are involved, differences in degradant profiles and other characteristics (e.g., rate constants, apparent reaction order) in the amorphous-state may simply reflect altered rates for individual reaction steps due to glass-induced changes in relative reactant mobilities rather than a change in overall mechanism.     

柔红霉素毫微粒冻干针剂的研究

目的：制备易再分散、稳定的柔红霉素聚氰基丙烯酸正丁酯毫微粒（ＤＮＲ－ＰＢＣＡ－ＮＰ）冻干针剂。方法：选用适宜支架剂制得ＤＮＲ－ＰＢＣＡ－ＮＰ冻干针剂,并评价其相关理化性质。结果：冻干前后毫微粒形态、粒径、ｐＨ、包封率及载药量均无明显变化,含水量合格,再分散性良好,制剂稳定。其临界相对湿度为７５．３３％。结论：在适宜的处方及工艺条件下制备ＤＮＲ－ＰＢＣＡ－ＮＰ冻干针剂是可行的。     

The Use of Lyophilized Vein Grafts in Vascular Access for Chronic Hemodialysis

The feasibility of utilizing lyophilized veins for creation of A-V fistulas was examined in two studies. In Study I, the longevity of lyophilized vein fistulas was found to be comparable to that of fresh veins in the experimental animal. Study II showed that lyophilized cadaveric veins performed at least as well as bovine grafts when used for vascular access in chronic hemodialysis patients. The advantages of lyophilized veins for creation of A-V fistulas in dialysis patients are described.     

Lyophilized Lecithin Based Oil-Water Microemulsions as a New and Low Toxic Delivery System for Amphotericin B

Purpose. To develop and investigate lecithin based oil-water microemulsions as potential amphotericin B (AmB) delivery systems and to evaluate their in vivo acute toxicity. Methods. AmB was added to the microemulsion and its location was evaluated by partitioning studies and UV-visible spectrophotometric analysis of the drug. Both, non-lyophilized and reconstituted microemulsions were characterised and assessed for their stability. Single-dose acute toxicity of the AmB microemulsion was studied on male albino Webster-derived CD-1 mice and compared with Fungizone®. Results. The studies performed showed that AmB was intercalated on the oil-water interface of the microemulsion as a complex formed with lecithin molecules. AmB addition did not seem to modify the rheological properties of the original system, but had an effect on its particle size distribution. Lyophilization of the microemulsion led to an oily cake, easily reconstituted and stable at the conditions studied. Single-dose acute toxicity studies proved that the LD₅₀ of AmB microemulsions was of 4 mg kg^–1 of animal weight, compared with 1 mg kg^–1 found for Fungizone®. Conclusions. Lyophilized lecithin based oil-water microemulsions appear to be valuable systems for the delivery of AmB in terms of easy and low-cost manufacturing, stability and safety compared with the formulations already in market.