注射用重组新蛭素的质量控制研究

目的:建立注射用重组新蛭素原液、成品的质量控制方法及内控质量标准。方法:利用纤维蛋白凝块法测定注射用重组新蛭素的生物学活性,非还原SDS-PAGE和RP-HPLC测定纯度,SDS-PAGE和质谱法分析相对分子质量,免疫印迹验证目的蛋白,其余检测项目按《中华人民共和国药典》(2010年版三部)规定进行。结果:用建立的方法对注射用重组新蛭素3批原液和成品进行了检定,原液抗凝比活性均为512 ATU/mg,非还原型SDS-PAGE和HPLC检测3批原液的纯度结果均大于95%;质谱法分析3批原液相对分子质量符合7.3×103±0.7×103,SDS-PAGE分析3批原液相对分子质量符合13.2×103±1.3×103;免疫印迹表明检测条带为目的蛋白,能被抗水蛭素抗体特异性结合;其余各项指标均符合内控质量标准及《中华人民共和国药典》(2010年版三部)的要求。结论:建立的质量控制方法和内控质量标准可以用于注射用重组新蛭素的检定和质量控制。     

重组定点突变巴曲酶质量标准研究

目的 建立重组定点突变巴曲酶的质控方法和质量标准.方法 生物学活性测定采用血浆凝集活性测定法;通过胰蛋白酶消化和RP-HPLC法分析该蛋白还原型肽图;其余检测项目均按<中华人民共和国药典>2010年版(三部)相关规定进行.结果 用建立的方法对三批重组定点突变巴曲酶原液和成品进行检定,各项指标均符合2010版<中华人民共和国药典>和相应指导原则的要求.三批原液比活性均≥2000 kU/mg.肽图三批次之间一致,原液的蛋白含量、纯度、分子质量、等电点、N末端氨基酸序列等指标均符合规定.结论 建立的质控方法可有效地控制重组定点突变巴曲酶产品质量,并可用于定点突变巴曲酶原液及成品的常规检定.     

关于我国药典重组DNA技术产品总论的思考

自1982年全球第一个生物技术药物“基因重组人胰岛素”、1989年中国批准第一个生物技术药物“重组人干扰素α1b”上市以来,生物技术药物已成为制药业中发展最快、活力最强和技术含量最高的领域。药品的规范生产与质量控制与其安全有效性密切相关,欧美药典中均设有对此类药品质量控制的总体要求。《中国药典》2010版三部已收录包括12类共计34个品种的重组DNA技术产品各论,在进一步保障药品安全、提高质量控制水平的编制指导思想下,《中国药典》2015版拟纳入对重组DNA技术产品的总体要求,本文就相关起草工作从产品涉及范畴、制造与产品检定等方面进行阐述。     

PEG化重组人白细胞介素-6制品游离PEG测定方法的研究

为了建立PEG化学修饰的重组人白细胞介素-6(PEG-rhIL-6)的部分质量控制方法,参照2005年版《中华人民共和国药典》三部附录III B高效液相色谱法,以RP-HPLC方法分离PEG-rhIL-6原液中的不同成分,用蒸发光散射监测器检测游离PEG,用外标法测定计算样品中残留PEG的含量。PEG修饰rhIL-6结构稳定;制品中的游离PEG含量符合要求。RP-HPLC法检测游离PEG结果可靠。     

甲型H1N1流感疫苗生产用工作毒种的质量控制

根据中国药典2005年版三部和WHO"人用大流行流感疫苗制备的指导原则"相关要求,以及各企业的申报规程,对全国10家甲型H1N1流感疫苗生产企业工作毒种A/Californ ia/07/2009 NYMC X-179A进行毒种检定,结果均符合中国药典2005年版三部和各企业申报规程的要求。     

重组人戊型肝炎疫苗细菌内毒素检查方法的建立

确立重组人戊型肝炎疫苗原液的细菌内毒素检查方法。供试品参照《中华人民共和国药典》(2005年版三部)热原检查法进行热原检查,结果符合规定。该供试品同时参照《中华人民共和国药典》(2005年版三部)细菌内毒素检查法要求进行试验。供试品溶液在40μg/m l浓度下,确定内毒素限值为40EU/m l。供试品在该内毒素限值下干扰试验有效,且细菌内毒素检查法符合规定。该疫苗用内毒素检查法代替热原检查法,方法可行。     

重组人促红细胞生成素在国内外药典中质量标准不同点的对比分析

目的:对比重组人促红细胞生成素在《欧洲药典》(第5版)和《中国药典》(2005年版)中标准的不同点,为国内研发机构对基因重组蛋白产品的研发及国内企业对该产品的进出口提供参考。方法:按2部药典要求,对进口产品或国产产品实样进行部分关键指标的对比分析。结果:2部药典对该产品的标准描述上存在具体内容的区别。结论:在现代药物的研发和生产中,对药品的国际及国内标准要进行综合分析而拟定。     

重组人干扰素注射剂制造浅析

干扰素注射剂是历版《中国药典》收载的最主要的一类重组技术制品。对于生物制品而言,生产过程与制品,制品的规范生产与其安全性、有效性密切相关。剖析了重组人干扰素注射剂规程中对制品制造的要求,包括基本要求以及菌种库建立、原液制备、半成品制备、成品制备等环节;历版药典对制造要求的演变过程,并与《欧洲药典》、《美国药典》进行了对比。介绍了2015年版《中国药典》拟增订的"重组技术制品总论",该总论对于已上市品种的规范生产、新品种研发都具有指导意义。"重组技术制品总论"是对该类制品生产检定提出的一般原则性要求,相关各论的起草均应以此为基础,并与之相互呼应协调。     

《中国药典》重组人干扰素注射剂质量标准浅析

本文剖析了现行版《中国药典》收载的重组人干扰素注射剂质量标准相关方法、检测限度和历史沿革;对比研究了与国外先进药典如欧洲药典的差距,包括相关物质、相关杂质分析、生物学活性测定结果的统计分析、比活性等方面;介绍了2015年版《中国药典》拟增修订主要内容,如增订报告基因法检测干扰素生物学活性、增订定量PCR法检测外源DNA残留量,加强"理化对照品"的管理,相关检定机构对国内生产企业的理化对照品进行了标定。本文还探讨了提高该类制品质量标准的主要方向。     

重组人白介素-11蛋白浓度两种测定方法比较

在基因工程蛋白药物开发研究过程中,选定恰当、准确的蛋白浓度测定方法非常重要。本文比较Lowry法和Bradford法测定重组人白介素-11蛋白浓度结果,认为选用Bradford法是可行的,但中国药典三部蛋白浓度测定方法中没有收录Bradford法,建议在中国药典三部修订时收录该方法。     

人降钙素基因相关肽脂质体质量标准的研究

建立完整的人降钙素基因相关肽脂质体(LipohCGRP)药物的质量标准。用家兔球睫膜血管扩张法测定hCGRP的生物学活性;采用RPHPLC、等电聚焦、薄层层析等方法分别测定样品的纯度、等电点和迁移率及脂质体嵌入率的测定,并按照中国生物制品规程的要求完成了甲醇和氯仿等残留物质的分析和动物安全试验。建立了活性测定方法,能准确测定样品中不到1ng的样品活性单位,连续三批样品的各项指标均符合质量标准的要求。建立了脂质体多肽药物的质量标准,并用于质量检定。     

Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea

Eucaryotic expression systems are widely used to produce biologicals for human use, e.g. vaccines, recombinant proteins and monoclonal antibodies. As part of the safety testing the current U.S. Food and Drug Administration (FDA) regulatory guidelines as well as several European Pharmacopoiea monographs requests the demonstration of the absence of Mycoplasma in the cell culture in the bioreactors prior to harvest and further downstream processing. In recent years progress has been made in the development of a sensitive NAT-based method for the detection of Mycoplasma species in CHO cells, e.g. Eldering et al. This method is based on a nucleic acid amplification technique using a very sensitive touch-down PCR-profile. The presence of mollicutes DNA in the test specimens is determined by an approx. 450 bp target sequence which is amplified and this amplicon is finally detected by polyacrylamide gel electrophoresis. Based on this method a ready-to-use test kit was developed. In this report the validation of both method variants according the European Pharmacopoiea monograph 2.6.7 “Mycoplasmas” is described. The validation demonstrated the robustness and precision as well as a sufficient specificity of both assay formats. The validated sensitivity fulfills the requirements of the European Pharmacopoiea for a PCR-based method proposed as an alternative to the time consuming indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).     

Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods

AIMS: The in vitro activity of some essential oils (EO) (thyme red, fennel, clove, pine, sage, lemon balm and lavender) against clinical and environmental fungal strains was determined. METHODS AND RESULTS: The minimal inhibitory concentrations were determined by a microdilution method in RPMI 1640 and by a vapour contact assay. The composition of oils was analysed by gas chromatography (GC) and GC/mass spectrometry. The results indicated that the oils antifungal activity depended on the experimental assay used. The inhibiting effects of EO in vapour phase were generally higher than those in liquid state. According to both methods thyme red and clove were found to be the oils with the widest spectrum of activity against all fungi tested. CONCLUSIONS: Despite the differences between the two methods, our results demonstrate that some EO are very active on dermatophytes and dematiaceous fungi. However, more data will be necessary to confirm this good in vitro efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: This study could identify candidates of EO for developing alternative methods to control environmental and clinically undesirable filamentous fungi.     

Validation of an automated method of endotoxin testing for use in the end-product testing of ex vivo activated T-lymphocytes used in a somatic cell therapy

The aim of this study was to compare two methods of endotoxin testing to optimize quality control testing methodologies required for the rapid and precise determination of pyrogenicity in cell or tissue products used in cellular therapies. An automated kinetic-chromogenic method for determination of endotoxin levels in ex vivo activated T-lymphocytes infusion product, has been validated following the FDA guideline for the Limulus amebocyte lysate (LAL) assay. The validation protocol included: (1) initial qualification of the laboratory conducting the assay; (2) inhibition and enhancement tests both for treated (heated at 70 degrees C for 10 min) and untreated specimens; and (3) comparison of this assay with the conventional gel-clot method. Inhibition and enhancement testing showed that a 1:30 dilution of the infusion product was the optimal dilution for this type of specimen. Heating specimens did not appear to provide any advantage. A comparison study was performed on 105 infusion product samples. Endotoxin levels determined by both methods for all samples tested were within established end-product release specifications. The K-QCL method can be effectively used for endotoxin determination of ex vivo activated T-cells. (c) 1996 John Wiley & Sons, Inc.     

Heterologous expression of bovine lactoferricin in <Emphasis Type="Italic">Pichia methanolica</Emphasis>

According to the bias of codon utilization of Pichia methanolica, a fragment encoding bovine lactoferricin has been cloned and expressed in the P. methanolica under the control of the alcohol oxidase promoter, which was followed by the Saccharomyces cerevisiae alpha-factor signal peptide. The alpha-factor signal peptide efficiently directed the secretion of bovine lactoferricin from the recombinant yeast cell. The recombinant bovine lactoferricin appears to be successfully expressed, as it displays antibacterial activity (antibacterial assay). Moreover, the identity of the recombinant product was estimated by Tricine-SDS-PAGE.     

重组双功能水蛭素的发酵、纯化和鉴定

构建了重组双功能水蛭素 ( Recombinant-RGD-Hirudin、 r-RGD-Hirudin ) cDNA 的表达质粒 RGD-Hirudin-pPIC9K,转化入毕赤酵母中,经筛选得到高表达的阳性克隆。种子菌经过3d发酵培养,其培养液上清经超滤浓缩、凝胶过滤层析和离子交换层析后,得到纯度大于97%、比活性为 12000 ATU/mg 的 r-RGD-Hirudin,回收率大于60%,发酵产率为 1 g/L。纯化后的 r-RGD-Hirudin 经过还原SDS-PAGE,抗凝血酶活力分析、抗血小板聚集分析、质谱分析及等电聚焦分析等方法鉴定,证明该表达产物为水蛭素的衍生物,具有抗凝血酶和抗血小板聚集双重功能。     

Use of an ATP assay to determine viable microbial biomass in Fennoscandian Shield groundwater from depths of 3-1000 m

Bioluminescent biomass in pure cultures and in 94 samples of shallow and deep Fennoscandian Shield groundwater was analysed using a commercial ATP assay, and the results were compared with microscopic counts and counts based on cultivation methods. The assay appeared robust and reliable and had a detection range that covered all samples analysed. The detection limit in groundwater was determined to 2x10(3) cells ml(-1). ATP concentrations were found to correlate with the microscopic counts and with the volume and metabolic status of the investigated pure culture and groundwater cells. The results suggested that bacterial populations in deep groundwater vary significantly in size, and that metabolic activity is a function of prevailing environmental conditions. In cases in which analysis of the total and viable number of cells produced very low numbers, suggesting that the detected cells were of low viability, ATP analysis of the ratio of ATP to the total number of cells was able to verify such an interpretation of the obtained data.     

Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia.

Cordyceps, one of the well-known traditional Chinese medicines, consists of the dried fungus Cordyceps sinensis growing on the larva of the caterpillar. It is commonly used for the replenishment of body health. One of the known pharmacological effects is its anti-oxidation activity. However, there is a great variation of the quality in different sources of Cordyceps. Here, the water extracts of various sources of natural C. sinensis and cultured Cordyceps mycelia were analyzed for their anti-oxidation activity by using three different assay methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. The results showed that Cordyceps, in general, possesses a strong anti-oxidation activity in all assays tested. However, both natural and cultured Cordyceps showed the lowest inhibition in the lipid peroxidation when compared with the other two assay methods. The cultured Cordyceps mycelia had equally strong anti-oxidation activity as compared to the natural Cordyceps. Besides, the anti-oxidation activities were increased to 10-30 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from Cordyceps polysaccharides.     

The activity of S9-liver fractions from seven species in the Salmonella/mammalian-microsome mutagenicity test

A comparative study was made of the metabolizing activity of microsomal fractions of the liver of the rat, mouse, Chinese hamster, dog, mini-pig, rhesus monkey and baboon, with and without pretreatment of the animals with Aroclor (500 mg/kg i.p.). The activity of the fractions was determined by means of the Salmonella/mammalian-microsome mutagenicity test. The protein content of the S9 fractions was standardized (36.14 mg/ml) to eliminate species-specific and inter-individual differences. Strain TA100 of Salmonella typhimurium was used as test organism. The substances tested, namely benzo[a]pyrene, cyclophosphamide, diethylnitrosamine, β-naphthylamine and o-aminoazotoluene, are all known mutagens or carcinogens belonging to various chemical classes.

The tests made with S9 fractions from animals that had not been pretreated with Aroclor revealed distinct species-specific differences in the metabolizing activity of the fractions and also differences from one substance to another. The fractions from mice and Chinese hamster had only a weakly positive effect with some of the substances, those from the rat and the mini-pig only with 2 and 3 of the 5, resp. The dog-liver fraction gave positive results with all substances except benzo[a]pyrene. The fractions from the baboon and the rhesus monkey had positive to strongly positive effects with all the substances.

In the experiments in which the animals had been pretreated with Aroclor, the S9 fractions from all species produced positive effects with all substances, and the mutagenic effects provoked by various substances, when fractions from untreated animals were used, remained of the same degree or became more distinct. Species-specific differences were no longer discernible.

Demethylase activity in all the S9 fractions used was determined with ethylmorphine. Without prior activation, the fractions from the mouse, Chinese hamster, rat and dog gave highly divergent values. In the fractions from the mini-pig, the baboon and, in particular, the rhesus monkey, the activity was relatively more pronounced. Enzyme induction with Aroclor led to a distinct increase in demethylase activity in the livers of almost all species. Only a very rough quantitative relation was evident between the demethylase activity of the individual S9 fractions and their influence on the substances tested for mutagenicity.     

Expert examination of the immunogenic activity and toxicity of the pertussis component of adsorbed and non-adsorbed diphtheria-tetanus-pertussis vaccines. Results of interlaboratory investigations under the WHO project "Reactogenicity and Toxicity of DPT Vaccines"

Since the DPT vaccine is broadly used for the prevention of diphtheria, tetanus and pertussis' its preparations should meet special quality requirements. The present study was aimed at evaluating the reproducibility of laboratory methods utilized to assess the protective activity and toxicity of the pertussis component as well as at examining the feasibility of expert estimations of product quality to enhance the validity of findings. The results of interlaboratory comparative examination of quality parameters of the tested preparations revealed that the routine laboratory quality control methods were not sufficiently standardized as their application in different laboratories did not always produce identical results. The WHO criteria to evaluate the toxicity of pertussis vaccines are far from perfect since vaccines with pronounced toxicity can only be distinguished from those with moderate to mild toxicity after the administration to tested mice of vaccines at a single infant dose of 0.5 ml. To enhance the reproducibility of methods employed in the laboratory, appropriate standard specimens of the preparation should be used serving as a measure of different quality parameters; all test conditions should be also standardized as far as possible. To enable objective quality evaluation of medical biological preparations, the degree of experiment reproducibility should be regularly verified in interlaboratory tests on an international scale as well as inside those countries which have several manufacturers of a given preparation. It appears expedient to set up an international system of expert evaluation of quality of biological preparations by appointing several regional and national centres which meet the requirements for expert laboratories.