Changes in the cell size of Brevundimonas diminuta using different growth agitation rates

Brevundimonas diminuta (ATCC 19146) is a standard organism for validation of sterilizing-grade membrane filters. Cell size is critical for the determination of retention characteristics of 0.2 micron rated membrane filters. In this study, cell size changes of B. diminuta cultured under different physiologic states and variable agitations at 50, 100 and 200 rpm were measured by a particle size analyzer and scanning electron microscope (SEM). The smallest cells were obtained at initial stationary phase in saline lactose broth (SLB) as a shaking culture at 50 rpm. Cells grown under agitation at 50, 100 and 200 rpm showed an increase of specific growth rate (mu), about 2.9, 3.6 and 3.6 fold, respectively, compared to the non-agitated cells in SLB media. These results suggested that the cell size decreased proportionally with increase of the specific growth rate (mu) in SLB. These size changes were associated with penetration through a 0.2 micron rated cellulose acetate filter. A scale-down filtration system was developed and performed bacterial challenge test and bubble point test with cells cultured in SLB. Cells grown under agitation conditions in SLB were not retained by 0.2 micron rated membrane filter.     

The importance of accurate microorganism identification in microbial challenge tests of membrane filters--part I

Microbial challenge testing is a common procedure to determine the retention efficiency, performance, and validity of a sterilizing-grade filter. The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146), routinely used to verify a 0.2 μm rated sterilizing-grade filter. Process validation procedures most often also utilize B. diminuta (ATCC 19146), but instead of the standard procedures and fluids, process, and product parameters are employed to determine whether these parameters influence the retentivity of the filter or changes to the challenge organism, which might result in the penetration of the filter. In certain instances, the native bioburden within the drug manufacturing process is used to perform such process validation challenge tests. Filter penetrations can happen and cause concern; therefore, it is essential to identify the organism species with accuracy to avoid unnecessary confusion. This paper and its follow-up will describe such imprecision and the resulting misconceptions. It will clarify past determinations and put perspective on the findings. LAY ABSTRACT: Sterilizing-grade filters are used to remove microorganisms from biopharmaceutical solutions. To determine the retention performance of such filters, bacteria challenge tests are utilized, often with a standard challenge organism (Brevundimonas diminuta), in instances with native bioburden. The accuracy of the microorganism identification is of importance to avoid flawed results and misinterpretation of the filter's performance.     

Dose accuracy of a reusable insulin pen using a cartridge system with an integrated plunger mechanism

Pen injection devices are a common method of administering insulin for patients with diabetes. Pen devices must comply with guidelines prepared by the International Organization for Standardization, which include device dose accuracy and precision. OptiClik (sanofi-aventis) was developed to fulfil unmet needs of patients with diabetes, including: easier cartridge changing, clearer dose display and readability, and a larger dose of insulin to be delivered with a single injection. In this paper, the authors report on the dose accuracy of the OptiClik pen device, which uses a novel cartridge system with an integrated plunger for easier cartridge changing. The authors show that OptiClik accurately delivers a required dose of insulin, which is maintained over the lifetime of the pen. OptiClik offers a significant contribution to the treatment of diabetes.     

Extractables from filter components in model solvent streams for pharmaceutical filtration processes

Drug manufacturers use filters widely in their manufacturing processes for bioburden reduction and sterility assurance. In implementing a pharmaceutical filtration, it is essential to determine and profile the filter extractables that could be introduced into the product. There is an ongoing industry effort to explore the analysis of extractables. Toward this end, the use of complementary analytical techniques was evaluated to aid in extractables identification by determining whether the analysis of filters in the form of components, rather than entire assemblies, facilitated the profiling of extractables. The study examines filter components of 10-inch, 0.22-microm Durapore cartridge filters using Fourier transform infra-red spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR), as well as determines the non-volatile residues (NVR) and total organic carbon contents (TOC) of the extractables. The complementary techniques of high performance liquid chromatography (HPLC/UV) and liquid chromatography nuclear magnetic resonance spectroscopy (LC/NMR) were also used. The results of the tests indicated very low levels of extractables.     

In vitro performance characteristics of valved holding chamber and spacer devices with a fluticasone metered-dose inhaler

STUDY OBJECTIVE: To compare the in vitro aerosol deposition characteristics of several commercially available valved holding chamber (VHC) and spacer devices used with a fluticasone metered-dose inhaler (MDI). DESIGN: In vitro aerosol deposition study SETTING: University-affiliated research center. DEVICES: Seven VHC devices: BreatheRite, E-Z Spacer, EasiVent, AeroChamber, InspirEase, OptiChamber, and Space Chamber. Six spacer devices: OptiHaler, Aerosol Cloud Enhancer (ACE), Gentle-Haler, MediSpacer, Ellipse, and a 6-inch tube (1-inch inside diameter). INTERVENTION: The respirable dose (aerosol particles 1-5 microm) of fluticasone was determined by sampling 10 220-microg actuations from five runs with each spacer or VHC plus MDI combination, by using a well-established in vitro cascade impactor method. MEASUREMENTS AND MAIN RESULTS: Fluticasone aerosol was washed from the impactor with methanol and quantified by means of high-performance liquid chromatography. Differences among outcomes were determined with analysis-of-variance testing. Among spacers, Ellipse had the highest respirable dose (104 microg, p < 0.01). Respirable doses for the 6-inch tube (74.3 microg), Gentle-Haler (81.7 microg), and MediSpacer (82.6 microg) were no different from that of the MDI (p > 0.05), whereas respirable doses of OptiHaler (44.6 microg) and ACE (47.2 microg) were less than those of all other spacers (p < 0.001). Among VHC devices, respirable doses from EasiVent (35.6 microg), AeroChamber (47.0 microg), InspirEase (52.7 microg), OptiChamber (53.1 microg), and Space Chamber (58.3 microg) were not different (p > 0.05), whereas BreatheRite (13.1 microg) and E-Z Spacer (27.3 microg) respirable doses were less than those of the other VHC devices (p < 0.05). CONCLUSION: Spacers and VHC devices available in the United States do not demonstrate equivalent in vitro performance with the fluticasone MDI. The difference between highest and lowest respirable doses in each device category would likely lead to clinically relevant differences in the quantity of fluticasone delivered to a patient.     

角膜基质内植入改性聚羟乙基丙烯酸甲脂材料的生物学反应

目的评价角膜层间植入改性聚羟乙基丙烯酸甲酯(Poly(2-hydroxyethyl methacrylate,PHEMA)孔隙性材料的生物学反应。方法选用20只新西兰白兔,将PHEMA材料植入角膜基质内,定期观察。结果植入后PHEMA材料全部稳定存留于角膜内,角膜组织呈穿通性长入PHEMA材料孔隙内,现最长达12个月以上。未见PHEMA材料排出,也未发生白内障、视网膜脱离、角膜后膜等并发症。术后并发症包括睑缘红肿、畏光、流泪等刺激症状,均于术后1周内消失；组织学提示PHEMA材料孔隙内有多量的成纤维细胞并有胶原等细胞外基质沉积。电镜检查PHEMA材料周围角膜细胞功能活跃,分泌胶原,类似组织愈合过程。结论PHEMA孔隙材料是人工角膜支架的理想材料；由于该材料还可以制作成光学性能良好的光学部,故可望制成一体式人工角膜。     

Impact of tubing material on the failure of product-specific bubble points of sterilizing-grade filters

The following study was conducted to determine the effect of different preservatives commonly used in the biopharmaceutical industry on the product-specific bubble point of sterilizing-grade filters when used to filter product processed with different types of tubing. The preservatives tested were 0.25% phenol, m-cresol, and benzyl alcohol. The tubing tested was Sani-Pure (platinum-cured silicone tubing), Versilic (peroxide-cured silicone tubing), C-Flex, Pharmed, and Cole-Parmer (BioPharm silicone tubing). The product-specific bubble point values of sterilizing grade filters were measured after the recirculation of product through the filter and tubing of different types of materials for a total contact time of 15 h. When silicone tubing was used, the post-recirculation product-specific bubble point was suppressed on average 13 psig when compared to the pre- recirculation product-specific bubble point. Suppression was also observed with C-Flex, but to a much lesser extent than with silicone tubing. Suppression was not observed with Pharmed or BioPharm tubing. Alcohol extractions performed on the filters that experienced suppressed bubble points followed by Fourier transform infrared spectroscopy analysis indicated the filters contained poly(dimethylsiloxane). Direct addition of poly(dimethlysiloxane) to solutions filtered through sterilizing-grade filters suppressed the filter bubble points when tested for integrity. Silicone oils most likely reduced the surface tension of the pores in the membrane, resulting in the ability of air (or nitrogen) to pass more freely through the membrane, causing suppressed bubble point test values. The results of these studies indicate that product-specific bubble point of a filter determined with only product may not reflect the true bubble point for preservative-containing products that are recirculated or contacted with certain tubing for 15 h or greater. In addition, tubing material placed in contact with products containing preservatives should be evaluated for impact to the product-specific bubble point when being utilized with sterilizing-grade filters.     

浅析我院基于不同用途的多功能水处理装置

目的:提高医院制剂用水的可靠性,改善全院伤病员饮水的质量。方法:我院与北京斯钛诺水技术开发有限公司联合研发了一套多功能水处理装置,并加以应用。结果与结论:该装置由原水处理系统、膜纯化系统、电解系统、储水系统、控制系统、罐装系统等6部分组成。其采用石英砂过滤、活性炭吸附、树脂软化、保安过滤进行预处理,使水质初步净化,再应用双级反渗透生成双级反渗透纯化水作为制药用水;或经纳滤后,再经碱性水电解机电解生成饮用的弱碱性小分子还原水,或者经酸化水电解机电解生成消毒用酸性氧化电位(消毒)水和清洗用强碱性去污水,满足了医院多方面的用水需求,使水源利用率基本达到100%,且水质均达到相关标准要求。     

Behaviour of polysorbate 20 during dialysis, concentration and filtration using membrane separation techniques

During formulation development of a therapeutic protein, combinations of buffers, pH and excipients need to be tested. As the protein bulk solution used for formulation development usually contains a buffer component at a defined pH and potentially one or more excipients already, this bulk requires to be processed. In case low concentrations of non-ionic surfactants, for example polysorbate 20, are already present in the bulk, the surfactant needs to be removed in lab-scale for further development use. The scope of the work was to study the behaviour of low concentrations of polysorbate 20 during membrane separation processes. The first part focuses on evaluating the behaviour of polysorbate 20 during a dialysis process, whereas the second part analyses concentration changes of polysorbate during a membrane concentration process using a stirred cell. The third part analyses potential membrane absorption of polysorbate at sterilizing-grade filters. In conclusion, it was found that polysorbate could not be significantly reduced during a dialysis process and accumulated during a membrane concentration process in unreproducable manner. During sterile filtration, no significant influence on the concentration of polysorbate was measurable. In any case, it is recommendable to quantify the concentration of polysorbate during critical membrane process steps in pharmaceutical industry.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part IV: Retention of hydrogenophaga pseudoflava (ATCC 700892) and Ralstonia pickettii (ATCC 700591) by 0.2 and 0.22 micron rated filters

Ralstonia pickettii has emerged as a bioburden microorganism of considerable importance in pharmaceutical processes utilizing conventional 0.2 or 0.22 micron rated "sterilizing grade" filters. In this article, we re-evaluated and studied the retention efficiencies of 0.2 micron rated nylon 6.6 and 0.22 microns rated modified polyvinylidene fluoride (PVDF) filters for Hydrogenophaga pseudoflava (ATCC 700892) and R. pickettii (ATCC 700591). Out of a total of forty-four 0.2/0.22 micron rated filters discs tested in this study (spanning different challenge fluids, different challenge conditions, and different filter types), H. pseudoflava penetration was observed for every filter disc tested. Log titer reduction (LTR) values ranged from 0.3 to 2.0 logs for 20-48 hour challenges conducted in Water for Injection (WFI), and 3.8-7.1 logs for 6-hour challenges conducted in Minimal Media Davis (MMD). For 0.2 micron nylon 6.6 filter discs, penetration by R. pickettii was observed only in WFI challenges and was dependent on the culture and challenge conditions used. Penetration by R. pickettii was also restricted to only those membrane discs that were very close to the filter manufacturer's production integrity test (the Quantitative Bubble Point, QBP, test) limit. Where R. pickettii penetration was observed, LTR values were significantly higher than those observed for H. pseudoflava with the same filter discs. This study: 1) supports the use of H. pseudoflava as a worst-case challenge model for R. pickettii in process- and product-specific bacterial retention testing; 2) provides experimental evidence, for the first time, for the need to include filter membrane lots that have a physical integrity test value at or near the filter manufacturer's production (lower) limit in these tests; and 3) demonstrates how a standardized membrane integrity test (such as the QBP test) can be used select such "worst-case" membranes and to verify the inclusion of such "worst-case" membranes in these tests, thus serving as the link between the membrane disc used in bacterial retention validation testing and the production process filter.     

A comprehensive analysis of the role of correction factors in the allometric predictivity of clearance from rat, dog, and monkey to humans

This study was conducted to comprehensively evaluate the performance of various allometric scaling methods for the prediction of human clearance. Allometric scaling was used to predict clearance for 103 compounds, for which clearance data in the rat, dog, monkey, and humans were available. Allometry was performed using all three preclinical species and with combinations of any two species. The methods employed included standard allometry and various correction factors, including brain weight, maximum lifespan potential, and glomerular filtration. Scaling was performed on all compounds universally and on segregated subsets based on allometric exponent, clearance, physicochemical property, or route of elimination. 776 allometric combinations with 27,313 individual outcomes were performed. A predicted-to-observed clearance ratio of 0.5 to twofold was preselected as the criterion for predictive success. The success rate of allometric scaling ranged from 18 to 53%; none of the correction factors resulted in substantially improved predictivity. Furthermore, none of the methods attempted in this study achieved a success rate greater than that observed by simply estimating human clearance based on monkey hepatic extraction. Prospective allometric scaling, with or without correction factors, represents a suboptimal technique for estimating human clearance based on in vivo preclinical data.     

Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters

Formulations of therapeutic proteins usually contain a surfactant such as polysorbate 80 to protect them against interfacial stresses. Since surfactants may interact with surfaces, the aim of the present work was to study the adsorption behavior of low concentrations of polysorbate 80 and of a monoclonal antibody during sterile filtration. Lab-scale tests were performed to study the adsorption behavior of a monoclonal antibody to different filter materials (PVDF, PES, CA, and Nylon) from different suppliers. Subsequently, protein and polysorbate 80 adsorption were tested in manufacturing scale experiments. It was found that the extent of protein adsorption differed with filter materials, but also with different suppliers. Prominently, Nylon filters showed the highest degree of protein adsorption. In manufacturing-scale filtration experiments, significant adsorption of polysorbate 80 to sterilizing-grade filters was found. Thus, the adsorption of both protein and polysorbate to filters should be taken into consideration in the formulation and manufacturing process and assessed on a case-by-case basis depending on the manufacturing process set-up.     

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.     

2型糖尿病血清β2-微球蛋白的临床意义

目的探讨2型糖尿病与血清β2-微球蛋白的关系。方法采用放射免疫分析法对30例正常人,72例2型糖尿病患者（其中早期糖尿病肾病35例,简写为DN,糖尿病未出现肾损害37例,简写为NDN）进行血清β2-微球蛋白测定,并分组进行分析。结果DN组血清β2-微球蛋白含量明显高于对照组,P〈0．01,差异有统计学意义。对照组、NDN组及DN组血清β2-微球蛋白的含量逐渐递增,NDN组与对照组相比,P〉0．05,差异无统计学意义。结论血清β2-微球蛋白在DN组明显升高,提示该指标对预测早期糖尿病肾病有重要意义     

Current industrial practices of assessing permeability and P-glycoprotein interaction

Combination of the in vitro models that are high throughput but less predictive and the in vivo models that are low throughput but more predictive is used effectively to evaluate the intestinal permeability and transport characteristics of a large number of drug candidates during lead selection and lead optimization processes. Parallel artificial membrane permeability assay and Caco-2 cells are the most frequently used in vitro models to assess intestinal permeability. The popularity of these models stems from their potential for high throughput, cost effectiveness, and adequate predictability of absorption potential in humans. However, several caveats associated with these models (eg, poor predictability for transporter-mediated and paracellularly absorbed compounds, significant nonspecific binding to cells/devices leading to poor recovery, variability associated with experimental factors) need to be considered carefully to realize their full potential. P-glycoprotein, among other pharmaceutically relevant transporters, has been well demonstrated to be the major determinant of drug disposition. The review article presents an objective analysis of the permeability and transporter models currently being used in the pharmaceutical industry and could help guide the discovery scientists in implementing these models in an optimal fashion.     

Effects of sterilizing-grade filters on the physico-chemical properties of onion-like vesicles

Spherulites are new promising multilamellar vesicles that we study in a drug delivery context. The sterilization of spherulites suspensions is a necessary step before biological tests and later, before pharmaceutical applications (for example, parenteral or local injections). Among all sterilizing operations, the filtration through 0.22 microm sterilizing-grade filters (of the type Millex (? 4 mm) by Millipore) is easy and rapid, and we decided to study it as a mean to obtain sterile suspensions. The spherulites diameter is usually comprised between 0.2 and 0.5 microm but bigger vesicles occur and reach ? 1 microm. The effects of such filters on vesicles' size and lipids' concentration were then compromised. After examination of this challenging operation, results proved that the sterilizing filtration had no effect on these two parameters whatever the formulation chosen. Then, the possible release of amaranth, an encapsulated hydrophilic dye was followed. With the formulations and in spite of a filter diameter inferior to that of the vesicles, the encapsulation yields were not significantly different before and after the filtration and no leakage could be detected. Finally, the spherulites' functionality after sterilizing filtration was studied under the chemical angle: vesicles containing an amphiphilic reactive anchor (CholE3ONH2) were still able to bind covalently a peptidic molecular recognition pattern. The ligation was quantified by fluorimetry as high as for non-filtrated suspensions. Thus, though spherulites can present a diameter superior to that of the sterilizing filters, their passage through them do not alter the physico-chemical properties of these vesicles.     

鼻腔喷雾剂给药装置的性能考察

目的:考察所选鼻腔喷雾剂给药装置的性能,建立评价该类装置性能的方法。方法:参考2010年版《中国药典》喷雾剂项下有关要求,分别以失重率和增重率为指标考察高温(60℃)和高湿(95%)条件下装置的密闭性,以及室温、40℃、60℃条件下的每喷喷量。结果:该装置在高温和高湿条件下失重率和增重率均<1%,3种条件下以60℃时的每喷喷量不准确、差异较大。结论:该装置的密闭性良好,在40℃及以下温度环境中放置时的每喷喷量准确、精密度好,符合鼻腔给药装置的要求。本文建立的方法可用于该类装置的性能考察。     

Electronic cigarette liquid and device parameters and aerosol characteristics: A survey of regular users

IntroductionElectronic cigarettes are widely variable devices, typically with user definable liquid and device parameters. Yet, little is known about how regular users manipulate these parameters. There is also limited understanding of what factors drive electronic cigarette use and liquid purchasing, and whether two common ingredients, propylene glycol and vegetable glycerin, alter the subjective effects of these devices.MethodsDuring the spring of 2016 522 adults, who reported daily use of electronic cigarettes containing nicotine, completed a survey on electronic cigarettes. Survey questions included an electronic cigarette dependence questionnaire, questions on tobacco and electronic cigarette use, and device and liquid preferences.ResultsFifty-nine percent of respondents reported using another tobacco product, which was positively associated with level of nicotine dependence. On average, devices were set to 28.3 (SD = 24.2) watts. Ability to change device voltage, and level of resistance typically used, was significantly associated with level of nicotine dependence. Amount of liquid consumed, nicotine concentration, and milligrams of nicotine used per week, were positively associated with nicotine dependence. Participants rated ‘good taste’ as the most important consideration when using and purchasing liquids, and propylene glycol is associated with undesirable effects and vegetable glycerin with desirable effects.ConclusionsThese data indicate that electronic cigarette users utilize a wide range device parameter settings and liquid variables, and that individuals with greater nicotine dependence favor voltage control devices, and lower resistance heating elements. Taste is a key factor for electronic cigarette selection, and concentrations of propylene glycol and vegetable glycerin may have a significant impact on the reinforcing effects of liquids.     

Immunocompatibility and biocompatibility of cell delivery systems

Immunoisolation therapy overcomes important disadvantages of implanting free cells. By mechanically blocking immune attacks, synthetic membranes around grafted cells should obviate the need for immunosuppression. The membrane used for encapsulation must be biocompatible and immunocompatible to the recipient and also to the encapsulated graft. The ability of the host to accept the implanted graft depends not only on the material used for encapsulation, but also on the defense reaction of the recipient, which is very individual. Such a reaction usually starts as absorption of cell-adhesive proteins, immunoglobulins, complement components, growth factors and some other proteins on the surface of the device. The absorption of proteins is difficult to avoid, but the amount and specificity of absorbed proteins can be controlled to some extent by selection and modification of the device material. If the adsorption of proteins to the surface of the implanted material is reduced, the overgrowth of the device with fibroblast-like and macrophage-like cells is also reduced. Cell adhesion at the surface of the implanted device is, in addition to the selected polymeric material, greatly influenced by the device content. Xenografts trigger a more vigorous inflammatory reaction than allografts, most probably due to the release of antigenic products from encapsulated deteriorated and dying cells which diffuse through the membrane and activate adhering immune cells. There is an evident effect of autoimmune status on the fate of the encapsulated graft. While encapsulated xenogeneic islets readily reverse streptozotocin-induced diabetes in mice, the same xenografts are short-functioning in NOD autoimmune diabetes-prone mice. Autoantibodies, to which most devices are impermeable, are not involved. Among the cytotoxic factors which are responsible for the limited survival of the encapsulated graft the most important are cytokines and perhaps some other low-molecular-weight factors released by activated macrophages at the surface of the encapsulating membrane.     

Development of a vacuum filtration system for the conventional microplate washer

INTRODUCTION: Filter plates are available from many vendors reflecting the growth of their scientific applications in various fields, however, the heterogeneous nature of those applications are the major factor to block the expansion of various filter plate options in a research scale operation. The development of an automatic vacuum filtration system for a conventional plate washer was presented as a possible solution for the filtration process in filter plate applications, especially in the melanin concentrating hormone receptor subtype 1 (MCH1) receptor binding assay with the time resolved fluorescence technology. METHODS: The pilot modification was done in the Embla 96/384 well washer by replacing the original plate carriage with a new carriage mimicking the conventional vacuum manifold to add the function of flow-through vacuum filtration for filter plates. The performance of new vacuum filtration system was evaluated with MCH1 receptor binding assay and ligand washout experiments. RESULTS: The mean background values from ligand washout experiments in AcroWell filter plates were 6406+/-502.9 with a manual vacuum manifold and 5563+/-585.8 with the vacuum filtration system. Z' factors were calculated as 0.6101+/-0.095 for the MCH1 receptor binding assay with the vacuum filtration system. DISCUSSION: The new plate carriage for a conventional plate washer was developed for filter plate applications to enable its use in a flow-through vacuum filtration application in addition to the conventional plate washing by an aspiration. The results from ligand washout and receptor binding assay suggest that the vacuum filtration system can provide a cost-effective solution for filter plate applications and may alleviate the most common problems of those heterogeneous assays to develop as high throughput operations without major investments for the professional workstations.