Obtaining higher transesterification rates with subtilisin Carlsberg in nonaqueous media

Three phase partitioning (protein precipitate obtained as an interfacial layer between lower aqueous and upper t-butanol phases, formed by the addition of ammonium sulphate and t-butanol to the aqueous solution of protein) followed by lyophilization in the presence of two-component excipient resulted in 400-480x increases in transesterification activity of lyophilized powders of subtilisin Carlsberg, depending on the solvent. The three phase partitioned enzyme, 'dried' by washing with butanol, gave 3-4x higher rates (depending on the solvent used) than the enzyme preparation dried by lyophilization in the presence of two-component excipient system.     

Modern Aspects of the Treatment of Hemophilia. III

In a previous article modifications were proposed in the preparation of the so-called two-donor fibrinogen, used in the treatment of patients suffering from hemophilia A. Only the freshly prepared product could be used, as lyophilization by the usual method caused varying, and at times considerable, losses in Factor VIII activity.In the present communication a method is described for lyophilization of the product by which the advantageous properties of the modified two-donor fibrinogen can be retained for a longer period. Factor VIII activity of this dried product was found to be nearly unchanged after storage for several months.     

仔猪副伤寒活疫苗中耐热保护剂应用相关参数的研究

【目的】研究并确定耐热保护剂在仔猪副伤寒活疫苗中应用的各参数,为制备细菌耐热保护剂活疫苗提供参考。【方法】将耐热保护剂与制苗菌液等体积混合,分别采用3种常用冻干曲线(1、2、3)进行冻干,抽样检测确定耐热保护剂配套冻干曲线。在确定合适冻干曲线的基础上,进一步研究耐热保护剂与不同菌龄菌液(发酵培养12、15、18和21 h)、不同浓度菌液(菌液终浓度分别为3×1010、5×1010和7×1010 CFU/m L)、不同感作时间(分别作用0、24和48 h)、耐热保护剂的不同保存时间(2-8°C分别保存0、10、20、30和40 d),以及不同分装量(2、3和4 m L)等多种不同参数对疫苗质量的影响。【结果】配套冻干曲线研究表明,曲线2冻干的产品性状、活菌存活率与耐老化指标效果最好;菌龄研究表明,37°C发酵培养18 h(位于对数生长末期或稳定前期),其冻干菌存活率达78%-81%,优于其它时间;配苗试验表明,菌液适宜终浓度为3×1010-5×1010 CFU/m L;最适感作时间为2-8°C感作24 h,冻干菌存活率可达85.3%-90.5%;耐热保护剂保存期试验表明,2-8°C保存40 d与0 d(配制当日)的保护剂冻干效果基本一致;配苗分装量试验表明,7 m L西林瓶中分装3 m L或4 m L,其冻干菌存活率与2 m L基本一致,但耐老化试验中活菌存活率比2 m L略高。【结论】收获发酵培养18 h的菌液、配苗终浓度采用3×1010-5×1010 CFU/m L,使用2-8°C保存40 d内的耐热保护剂,让保护剂与制苗用菌液2-8°C感作24 h,采用3-4 m L进行定量分装,按曲线2冻干为制备仔猪副伤寒耐热保护剂活疫苗的适合参数。     

Stabilization of purified potato virus X by dextran T-10 during lyophilization

Purification and preservation of potato virus X from leaf sap of tobacco plants before lyophilization was carried out by two methods: 1) precipitation by polyethylene glycol and ultracentifugation, and 2) precipitation by ammonium sulphate, chromatography on Sephadex G-50 and ultracentrifugation. The first method is preferable to the second because the final preparation contains more virus antigen. Both preparations were strongly infectious and maintained antigenic properties after lyophilization. To achieve a more gentle course of lyophilization of virus preparations, addition of urotropine and dextran T-10 to the virus suspension, purified by the precipitation by polyethylene glycol-6000, was examined. Addition of urotropine was proved unsatisfactory, because only antigenic properties were maintained after lyophilization while the infectivity disappeared. But we can recommend addition of dextran T-10 up to a concentration of 6% to the preparation of virus antigen before lyophilization. The course of lyophilization is much rapider, the lyophilized product can be very easily dissolved in water and is not hygroscopic. The product is strongly infectious and gives the serological precipitation reaction in a dilution four times that of X virus antigen lyophilized without addition of dextran T-10.     

Properties of live antibiotics-resistant anthrax vaccine STI-PR after long-term storage

Study showed that cultural, morphologic, genetic, immunologic characteristics, and resistance to antibiotics of STI-PR anthrax vaccine did not change after storage during 20 years in lyophilized condition. It has been shown that medium for lyophilization plays important role in preservation of vitality of anthrax spores. Optimal preservative properties have been observed for thioureal and sucrose-gelatinous media for lyophilization. Obtained results give reasons for prolongation of shelf live of STI-PR vaccine from 2 - 3 to 5 - 8 years.     

Dried nutrient media for the cultivation of pneumococcus and meningococcus

Dried synthetic nutrient medium for the cultivation of meningococci and the accumulation of their biomass has been developed. The kinetics of the culture growth, changes in pH and in the content of dissolved oxygen in the medium during prolonged controlled processes of the cultivation of meningococci in a bioreactor with the use of this medium have been studied. The stable physico-chemical properties and composition of the polysaccharide-protein complex isolated from the biomass of meningococci grown in the above-mentioned medium have made it possible to use it for the preparation of the samples of group B meningococcal vaccine. In addition, dried semi-synthetic nutrient medium for the accumulation of pneumococci without the necessity of introducing blood or serum into the medium has been developed. As regards its biological properties, this newly developed medium make it not inferior to meat media, containing blood or serum, and ensures good yield of biomass.     

Effect of Chemical Stabilizers on the Thermostability and Infectivity of a Representative Panel of Freeze Dried Viruses

As a partner of the European Virus Archive (EVA) FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20°C, +4°C and +20°C) for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID₅₀) assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses) still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.     

Long-term preservation of anammox bacteria

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.     

Biosorption of Copper(II) by Live and Dried Biomass of the White Rot Fungi Phanerochaete chrysosporium and Funalia trogii

Biosorption is an innovative and alternative technology to remove heavy metal pollutants from aqueous solution using live, inactive and dead biomasses such as algae, bacteria and fungi. In this study, live and dried biomass of Phanerochaete chrysosporium and Funalia trogii was applied as heavy metal adsorbent material. Biosorption of copper(II) cations in aqueous solution by live and dried biomass of Phanerochaete chrysosporium and Funalia trogii was investigated to study the effects of initial heavy metal concentration, pH, temperature, contact time, agitation rate and amount of fungus. Copper(II) was taken up quickly by fungal biomass (live or dried) during the first 15 min and the most important factor which affected the copper adsorption by live and dried biomass was the pH value. An initial pH of around 5.0 allowed for an optimum adsorption performance. Live biomass of two white rot fungi showed a high copper adsorption capacity compared with dried biomass. Copper(II) uptake was found to be independent of temperature in the range of 20–45 °C. The initial metal ion concentration (10–300 mg/L) significantly influenced the biosorption capacity of these fungi. The results indicate that a biosorption as high as 40–60 % by live and dried biomass can be obtained under optimum conditions.     

High yields of stable and highly pure nucleocapsid proteins of different hantaviruses can be generated in the yeast Saccharomyces cerevisiae

Recently, the high-level expression of authentic and hexahistidine (His)-tagged Puumala (strain Vranica/H?lln?s) hantavirus nucleocapsid protein derivatives in the yeast Saccharomyces cerevisiae has been reported [Dargeviciute et al., Vaccine, 20 (2002) 3523-3531]. Here we describe the expression of His-tagged nucleocapsid proteins of other Puumala virus strains (Sotkamo, Kazan) as well as Dobrava (strains Slovenia and Slovakia) and Hantaan (strain Fojnica) hantaviruses using the same system. All nucleocapsid proteins were expressed in the yeast S. cerevisiae at high levels. The nucleocapsid proteins can be easily purified by nickel chelate chromatography; the yield for all nucleocapsid proteins ranged from 0.5 to 1.5 mg per g wet weight of yeast cells. In general, long-term storage of all nucleocapsid proteins without degradation can be obtained by storage in PBS at -20 degrees C or lyophilization. The nucleocapsid protein of Puumala virus (strain Vranica/H?lln?s) was demonstrated to contain only traces of less than 10 pg nucleic acid contamination per 100 microg of protein. The yeast-expressed nucleocapsid proteins of Hantaan, Puumala and Dobrava viruses described here represent useful tools for serological hantavirus diagnostics and for vaccine development.     

Mixture of three amino acids as stabilizers replacing albumin in lyophilization of new third generation recombinant factor VIII GreenGene F

A formulation with stabilizers replacing albumin was developed for lyophilization of recombinant factor VIII (FVIII), GreenGene F (WHO INN: beroctocog alfa), to achieve stability and eliminate safety issues of blood‐derived albumin. L ‐Arginine (hydrophilic amino acid, positively charged side chain), L ‐glutamic acid (hydrophilic amino acid, negatively charged side chain), and L ‐isoleucine (hydrophobic amino acid, nonpolar) were selected as stabilizers, and the mixture of the three amino acids were optimized. The mixture had results comparative with albumin and other commonly used stabilizers showing good preservation of recombinant FVIII during lyophilization, robust stability with consistently high recovery of FVIIII, very low aggregate formation, and good storage stability without alterations in protein characteristics. In vivo test results showed that the efficacy was maintained and had no signs of toxicity. The study demonstrated that the three amino acid mixture acts as a good stabilizer for lyophilization of recombinant FVIII and as a safe excipient. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.     

The quality standardization of a national vaccine against yellow fever

One of the commercial lots of yellow fever vaccine has been attested as the National Branch Standard (NBS) of yellow fever vaccine. This NBS has been studied in all tests required by the regulations for standard vaccines. The NBS has been found to meet the necessary requirements in all its characteristics. The study of the thermostability of the NBS at temperatures of 4-10 degrees C, 20-22 degrees C, 37 degrees C during storage for 24 hours to 1 year has revealed the rapid loss of the infectious capacity of the virus at the above temperatures and its high stability during storage at -20 degrees C. Thus, the NBS has been found to retain the required level of immunizing potency for 3 months at a temperature of 4-10 degrees C, for 1 month at 20-22 degrees C and for 2 weeks (the term of observation) at 37 degrees C. The heat resistance of the NBS of yellow fever vaccine corresponds to the WHO requirements. The newly developed NBS has been used as the standard preparation for controlling 27 lots of commercial yellow fever vaccine.     

Viability of Bacillus popilliae after Lyophilization of Liquid Nitrogen Frozen Cells

The per cent viability of Bacillus popilliae after lyophilization of liquid nitrogen frozen cells was determined. Lyophilization of 9- to 12-hr cells which had been suspended in 5% sodium glutamate plus 0.5% gum tragacanth, frozen in liquid nitrogen vapor, and dried 4 to 5 hr with the ampoules exposed to room temperature resulted in survival of 64.6% of the original cells. After storage of these lyophilized preparations for 6 months at room temperature, 10.5% of the original cells were still viable.     

Effect of various procedures of storing the fungus Rhizopus microsporus UzLT-1, a lipase producer

The effect of various storage procedures on the viability and synthesis of lipolytic enzymes by Rhizopus microsporus UzLT-1 was studied. The best procedures of producer storage proved to be regular passages and lyophilization. These procedures made it possible to maintain stable activity of lipolytic enzymes produced by the fungus for a long time. The fungal storage in vaseline oil or in a dried state was less effective due to significant losses of its lipolytic capacity.     

不同干燥方法对蝇蛆蛋白粉营养价值的影响

采用化学分析和动物实验的方法对冷冻干燥、微波干燥和低温干燥的蝇蛆粉进行了营养评价。结果表明,3种干燥方法不影响蝇蛆粉的粗蛋白和灰分含量,但微波干燥蝇蛆粉的脂肪含量显著降低;氨基酸分析显示,冷冻干燥蝇蛆粉的氨基酸总含量>微波干燥的蝇蛆粉>低温干燥的蝇蛆粉,而3种方法干燥的蝇蛆粉EAA/TAA均等于或大于41%,说明干燥所得的蝇蛆粉均属优质蛋白。另外,与FAO/WHO参考模式值相比,冷冻干燥蝇蛆粉的必需和半必需氨基酸总量明显高于FAO/WHO参考模式值,微波干燥的蝇蛆粉与FAO/WHO参考模式值相当,而低温干燥的蝇蛆粉中必需与半必需氨基酸含量显著低于FAO/WHO参考模式值,说明微波干燥及低温干燥会破坏蝇蛆中的某些必需或半必需氨基酸。动物实验结果表明,冷冻干燥的蝇蛆粉在动物体内被吸收利用的程度最高,其次是酪蛋白,再次是低温干燥的蝇蛆粉,而微波干燥的蝇蛆粉被动物吸收利用的程度最低。     

Use of Acetone-Dried Vaccines for Preparing Capsular Antisera Against the Klebsiella Group and the Lyophilization of Klebsiella Cultures

Capsular antisera against the 72 types of Klebsiella have been prepared in rabbits by using an acetone-dried vaccine. It was shown that encapsulated cells dried with acetone and ground to a fine powder with a mortar and pestle retain their capsules. Antisera obtained from rabbits vaccinated with these vaccines had quellung titers ranging from 1:16 to 1:64. The dried vaccine was stable after storage for over 2 years at room temperature. Encapsulated cultures lyophilized in the presence of 5% sucrose, 5% sodium glutamate, and 5% polyvinylpyrrolidone remained viable and retained their capsules after storage for 10 months at room temperature.     

Vacuum foam drying for preservation of LaSota virus: Effect of additives

The purpose of this research was to apply vacuum foam drying (VFD) for processing of LaSota virus and to screen formulation additives for its stability. The aqueous dispersion of harvest containing sucrose or trehalose in combination with additive (monosaccharides, polymers, N-Z-amine) was prepared. The diluted dispersions in vials were vacuum concentrated, foamed to form a continuous structure, and vacuum dried. The products were evaluated for foam characteristics, residual moisture, virus titer, x-ray diffraction pattern, and stability profile. The foamability increased with solid content in solutions. The foamability of sucrose was enhanced with incorporation of N-Z-amine (10% and 15% wt/vol) and polyvinyl pyrrolidone (PVP K30, 3% wt/vol). The fructose- or galactose-containing mixtures were deposited irregularly on the vial surface. The virus titer increased with disaccharides in the formulation. Sucrose provided better protection than trehalose. Unlike lyophilization, N-Z-amine with sucrose protected the virus from Millard’s Browning. Amino acids do not have a catalytic effect on hydrolysis of sucrose during VFD. Monosaccharides were ineffective. A synergistic effect of PVP K30 or polyethylene glycol 6000 (3% wt/vol) with N-Z-amine provided the maximum virus titer (6.97 and 7.15, respectively). This formulation retained the desired virus potency at 5°, 25°, and 40°C. The diffraction pattern revealed that a threshold concentration of N-Z-amine was required for inhibiting crystallization of sucrose during VFD. VFD was successfully applied to produce a solid LaSota formulation. The products were amorphous and did not devitrify on storage. Published: July 21, 2006     

Application of accelerated storage test to lyophilized plant viruses

Summary The preservation of nine plant virus strains of tobamovirus and cucumovirus groups after freeze-drying in different lyophilic forms was examined. Quantitative studies on survival were performed. In tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), accelerated storage test at 70 – 100°C was applied for screening 20 protecting media. A perspective medium, 5% sorbitol, 3.6% dextran, for plant viruses lyophilization with high cryo- and xeroprotective effects was found.     

埃博拉病毒疫苗rVSV-ZEBOV的研究进展

埃博拉病毒被列为A类病原体,感染后可引起埃博拉出血热,具有高传染率和高致死率。研发安全有效的抗病毒疫苗迫在眉睫。目前正在研发的埃博拉病毒疫苗包括病毒载体疫苗、蛋白疫苗、DNA疫苗等,其中最有希望的是重组水疱性口炎病毒载体疫苗rVSV-ZEBOV。该疫苗在预防和治疗埃博拉出血热方面具有较高的安全性和有效性,有望在2018年上市。为了深入了解rVSV-ZEBOV疫苗,现主要从制备方法、药理学研究和作用机制等方面对该疫苗进行介绍。