麻疹、腮腺炎二联活疫苗的研制

用于生产麻诊活疫苗的“沪191”毒株和由武汉生物制品研究所引进的腮腺炎疫苗“Wms4”毒株生产各自的病毒原液,全面检定合格后,对麻疹与腮腺炎两种病毒原液进行配比试验、热稳定性试验等,最终选定联合疫苗中麻疹与腮腺炎病毒CCID50之比1：30,试制出二批麻－腮二联活疫苗,经全面检定,完全符现行规程要求,目前正在I期临床研究。     

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。     

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.     

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。     

冻干人用狂犬病纯化疫苗的研制

在以原代地鼠肾细胞培养生产狂犬病疫苗工艺的基础上,通过对病毒收获液的浓缩倍数、灭活方式的改进及优化、冻干稳定剂的筛选、冻干曲线的确立,制备出了冻干疫苗,其质量及稳定性较液体疫苗有了整体水平的提高,各项检定结果均符合《中国生物制品规程》(2000版)。     

高效价人巨细胞病毒免疫球蛋白的研制

目的 制备人源性高效价人巨细胞病毒免疫球蛋白(HCMV-IgC)。方法 用ELISA方法从检验合格的献血员血浆中筛选HCMV-IgC滴度≥1:5000的血浆,采用低温乙醇法制备HCMV-IgC。结果 按《中国生物制品规程2000版》肌肉注射人血球蛋白检定标准,各项检定均合格,且HCMV-IgC滴度为1:150000。结论成功地制备出高效价HCMV-IgC。     

流感病毒裂解疫苗裂解剂去除工艺改进及稳定性研究

目的改进流感病毒裂解疫苗裂解剂去除工艺,降低残余卵清蛋白和裂解剂含量,提高疫苗质量,降低成本。方法分别将A1、A3和B型流感病毒纯化液用磷酸缓冲液（PB）沉淀法去除裂解剂,经超滤、除菌制备原液,配制6批半成品,其中3批不含硫柳汞,3批含硫柳汞,经全面检定,并观察放置37℃、25℃和2～8℃不同时间的稳定性。结果该疫苗各项指标均符合《中国药典》（2010年版）三部要求,其中卵清蛋白平均为3．83ng／mL,裂解剂平均为57μg／mL,比改进前分别降低97．9％和69％。37℃放置4周、25℃3个月及2-8℃12个月后检定全部合格。结论该工艺步骤简单,去除卵清蛋白和裂解剂效果明显,是进一步提高疫苗质量和降低成本的有效工艺。     

无细胞百日咳工艺中PO4^3—影响DTaP免疫效力分析

用0.02mol/L磷酸盐缓冲液和不用磷酸盐缓冲液透析生产无细胞百日咳（aP）,按《2000年版生物制品规程》制备和检定氢氧化铝吸附无细胞百日咳、白喉、破伤风联合疫苗（DTaP）。发现用磷酸盐缓冲液和不用磷酸盐缓冲液透析生产aP组成DTaP免疫效力差异非常显著,后者100％（10／10）合格,前者合格率只有27％（3／11）,用钼兰法测定DTaP中PO4^3-离子浓度。结果表明后者的PO4^3-浓度只是前者的1／10,PO4^3-依次影响白喉、破伤风、百日咳免疫效价。     

生物物质无菌性基本要求(生物物质规程第六号)

前言 生物物质无菌性基本要求(生物物质规程第六号)系于1959年由世界卫生组织的一个研究组制订。这些基本要求对于所有不得污染微生物的生物制品都适用,并在迄今已行制订的所有单项生物物质的规程中俱已引用。第24次生物检定专家委员会同意:鉴于近来生物制品无菌试验方法的发展以及可供实用的检定方法的进展,无菌性基本要求应予修订,从而进行了此项工作。     

麻疹疫苗生产连续培养多次收获工艺研究

本研究对连续培养多次收获工艺用于麻疹疫苗生产的可行性、疫苗维持液保护剂、冻干保护剂及冻干过程等进行了大量反复试验,结果表明在现行条件下,本生产工艺具有重复性好、成本低、投入产出率高、易于质量控制等优势。对本工艺生产疫苗进行全面检定,表明成品滴度和稳定性试验指标等均高于９０版《生物制品规程》要求,并在试验基础上制定出了生产工艺流程。     

rhGM—CSF／LIF融合蛋白基因的克隆及表达

利用基因重组技术,人工构建了一个编码五肽Ｇ－Ｓ－Ｇ－Ｇ－Ｓ的基因接头,将ＧＭ－ＣＳＦ和ＬＩＦ的ｃＤＮＡ相连而构成融合基因,将融合基因载入原核表达载体ｐＢＶ２２０后转化大肠杆菌,经热诱导后进行Ｗｅｓｔｅｒｎ印迹反应鉴定证实获得ｒｈＧＭ－ＣＳＦ／ＬＩＦ融合蛋白（简称ｒｈｇＭ－ＬＩＦ）活性测定表明重组的融合蛋白具有两因子双重活性。     

The contribution from the choroid plexus and the periventricular CNS to amino acids and proteins in the human CSF

During neurosurgery the freshly secreted extracellular fluid (ECF) from the choroid plexus was sampled with small pieces of application paper in three patients with intractable epilepsy. The samples were analyzed for free amino acids and for soluble proteins. The results were compared with corresponding data on extracellular fluid from the brain surface obtained with dialysis-perfusion as well as with the cerebrospinal fluid (CSF) acquired by lumbar punction. The dialysis data were calibrated against the paper results. The choroid plexus secretion had a high concentration of transthyretin as well as of an unidentified protein with an isoelectric point of 7.4. The cortical ECF exhibited high concentrations of tau-globulin and gamma-trace protein. Among the amino acids, glutamine had lower concentration in the choroid plexus secretion and higher concentrations in the ECF of the brain compared to the CSF. The amino acid derivative ethanolamine exhibited a similar pattern. This was interpreted to demonstrate that these compounds enter the CSF from the brain tissue. In contrast, alanine, serine, and taurine had a lower concentration in the CSF than in the plexus secretion which suggests that they are removed from the CSF by brain tissue.     

Statistical assessment of DNA extraction reagent lot variability in real‐time quantitative PCR

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real‐time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot‐to‐lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.     

Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs

Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth. Quality control tools for FBS are relatively primitive and expensive given the complexity of the sample and the large amounts of FBS used. We undertook this study to examine whether proteomics could be used as a tool to analyze the variability of different fermentation processes. We hypothesized that inconsistent cell growth in fermentations could be due to the quality of FBS and that different lots of FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. To investigate whether this was the case, we grew three batches of adult retinal pigment epithelial cells (ARPE-19) using three different lots of fetal bovine serum (FBS-Ia, FBS-Ib, and FBS-II). We found that the growth rate of the culture was significantly and consistently higher in the FBS-II lot. To determine why the other lots promoted different growth properties, we used proteomic techniques to analyze the protein composition of the three lots. We then performed a time course study to monitor specific changes in individual proteins in the fermentation medium. The amount of several extracellular matrix and structural proteins, which are indicators of cell growth, increased over time. Alternatively, components supplied by the FBS addition, such as nutritional-related and cell-spreading-related proteins, decreased over time.     

Cytologic and immunocytochemical studies of cerebrospinal fluid in meningeal sarcoidosis. A case report.

Serial studies were done on cerebrospinal fluid (CSF) from a patient with sarcoidosis involving the meninges. Initially when the disease was active, the CSF protein was increased and glucose decreased. The number of cells in the CSF was moderately increased, and many mononuclear cells were present. Cytologic studies of the CSF showed many normal and some atypical lymphocytes. Immunochemical studies showed that most of these lymphocytes were T cells, with T-helper cells predominating over T-suppressor cells by a ratio of 3.92; B-lymphocytes were polyclonal. Subsequent studies of the CSF over the following three and one-half years showed that the protein and glucose content and the cell counts in the fluid did not correlate well with the activity of the disease. The number of atypical lymphocytes seemed to be a more useful marker of disease activity in the patient. Cytologic studies, when interpreted within the context of other CSF and clinical findings, are useful for the assessment of patients with sarcoidosis involving the meninges.     

Estimation of raw material performance in mammalian cell culture using near infrared spectra combined with chemometrics approaches

Understanding variability in raw materials and their impacts on product quality is of critical importance in the biopharmaceutical manufacturing processes. For this purpose, several spectroscopic techniques have been studied for raw material characterization, providing fast and nondestructive ways to measure quality of raw materials. However, investigations of correlation between spectra of raw materials and cell culture performance have been scarce due to their complexity and uncertainty. In this study, near-infrared spectra and bioassays of multiple soy hydrolysate lots manufactured by different vendors were analyzed using chemometrics approaches in order to address variability of raw materials as well as correlation between raw material properties and corresponding cell culture performance. Principal component analysis revealed that near-infrared spectra of different soy lots contain enough physicochemical information about soy hydrolysates to allow identification of lot-to-lot variability as well as vendor-to-vendor differences. The identified compositional variability was further analyzed in order to estimate cell growth and protein production of two mammalian cell lines under the condition of varying soy dosages using partial least square regression combined with optimal variable selection. The performance of the resulting models demonstrates the potential of near-infrared spectroscopy as a robust lot selection tool for raw materials while providing a biological link between chemical composition of raw materials and cell culture performance.     

Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) DNA was extracted from samples representing 10 lots of TM Biocontrol-1 stored at -10 degrees C for 5-15 years and digested with the restriction enzymes BglII, PstI, and SalI. DNA from the OpMNPV virus strain (MEM-75-STANDARD) used to produce the TM Biocontrol-1 lots was also extracted and digested. No restriction fragment length polymorphisms were observed in any of the samples and there was no evidence of DNA degradation. This indicates that long-term cold storage of TM Biocontrol-1 had no adverse effect on the quality of the OpMNPV DNA. In addition to the expected >23 kb OpMNPV DNA, extracts from lots 4a, 5b, and 6 contained 10 additional nucleic acid segments, ranging in size from 0.9 to 4.2 kb. The electrophoretic profile of these segments was characteristic of O. pseudotsugata cypovirus (OpCPV). RNase A/DNase I treatment showed that the nucleic acid contaminants were composed of RNA, suggesting that lots 4a, 5b, and 6 contained OpCPV as well as OpMNPV. Bioassay results have shown that there is a decrease in efficacy of stored TM-biocontrol-1, but this did not appear to be directly correlated with the length of time in storage.     

CSF-studies in neuropsychiatric disorders

Cerebrospinal fluid (CSF) is a clear and colourless fluid that surrounds the brain and spine. Due to the close proximity of CSF to the brain, pathological brain-processes are likely to be reflected in CSF. CSF can be obtained through lumbar puncture and is frequently performed in the differential diagnosis of neuropsychiatric disorders. Beyond clinical applications, CSF has been studied as part of different research-protocols. In this review, we will focus on CSF-analysis in Alzheimer Disease, major depression and schizophrenia. We will review both clinical applications as well as research applications in all three disorders. We will also assess new technological advances that have made it possible to study large numbers of proteins in CSF and how these advances may change CSF-analysis in the years to come.     

Seromonitoring of laboratory mouse and rat colonies for common murine pathogens (author's transl)]

During a period from 1973 to 1978, 392 and 225 lots including 12,232 mouse and 8,044 rat individual sera, respectively, were examined for antibodies to murine hepatitis virus, Sendai virus, Bordetella bronchiseptica, Mycoplasma pulmonis, Tyzzer agents, Salmonella typhimurium and Corynebacterium kutscheri. Of mouse lots 94.5% and 39.3% from breeder and user colonies, respectively, were negative for all antibodies examined as well as 31.6% and 17.2% of rat breeder and user colonies, respectively. Among positive lots from mouse users, high positivity rates were seen with Senai virus (47.6%), M. pulmonis (19.0%), and murine hepatitis virus (JHM : 18.2%, MHV : 31.0%), while the rates were high in rat user lots with Sendai virus (24.4%), B. bronchiseptica (39.3%) M. pulmonis (12.5%), murine coronaviruses (JHM : 19.0%, MHV-2 : 28.0%) and tyzzer agents (MSK : 19.6%, RT : 17.9%). These pathogenes with high positivities should be monitored indispensably as a quality control of laboratory mice and rats.     

Freezing of fresh Barhi dates for quality preservation during frozen storage

Fresh harvested dates are perishable and there is a need for extending their shelf life while preserving their fresh like quality characteristics. This study evaluates three different freezing methods, namely cryogenic freezing (CF) using liquid nitrogen; individual quick freezing (IQF) and conventional slow freezing (CSF) in preserving the quality and stability of dates during frozen storage. Fresh dates were frozen utilizing the three methods. The produced frozen dates were frozen stored for nine months. The color values, textural parameters, and nutrition qualities were measured for fresh dates before freezing and for the frozen dates every three months during the frozen storage. The frozen dates’ color values were affected by the freezing method and the frozen storage period. There are substantial differences in the quality of the frozen fruits in favor of cryogenic freezing followed by individual quick freezing compared to the conventional slow freezing. The results revealed large disparity among the times of freezing of the three methods. The freezing time accounted to 10 min for CF, and around 80 min for IQF, and 1800 min for CSF method.