重组人组织型纤溶酶原激活剂改构体质控方法的建立

目的建立重组人组织型纤溶酶原激活剂(tissue type plasminogen activator,tPA)改构体(TNK-tPA)的质控方法。方法采用气泡法测定TNK-tPA原液的生物学活性,非还原SDS-PAGE和RP-HPLC法测定其纯度,还原型SDS-PAGE确定其相对分子质量,HPLC-SEC法测定其单链含量,胰酶裂解法分析其肽图,Edman降解法测定其N-末端氨基酸序列,毛细管等电聚焦电泳法测定其等电点,Lowrry法测定其蛋白质含量,地高辛法测定其外源DNA残留量。按《中国药典》三部(2005版)要求,对TNK-tPA成品进行鉴别试验及外观、pH值、水分含量、无菌、装量、异常毒性等检测。结果 3批TNK-tPA原液的生物学活性为(2.09～2.16)×106U/ml,比活性均大于3.5×105U/mg,因此确定TNK-tPA原液的比活性不得低于3.0×105U/mg。3批TNK-tPA原液的非还原型SDS-PAGE纯度均大于98.0%,RP-HPLC纯度均大于97.0%;经还原型SDS-PAGE分析可见两条带,相对分子质量分别为68 000和35 000;样品原液的单链含量为75.7%;3批TNK-tPA原液的肽图酶切图谱与理化参考品一致;TNK-tPA原液的N-末端氨基酸序列、等电点、蛋白质含量、外源DNA残留量及成品的鉴别试验结果、外观、pH值、水分含量、无菌、装量、异常毒性等均符合《中国药典》三部(2005版)要求。结论建立的质控方法可保证产品安全、有效、质量可控,可用于TNK-tPA产品的常规检定。     

重组人骨形成蛋白-2的质量控制

目的建立重组人骨形成蛋白-2(rhBMP-2)质控方法和质量标准。方法以小鼠体内异位成骨试验测定rhBMP-2的生物学活性,HPLC-SEC法测定纯度,蛋白N-末端测序仪测定N-末端15个氨基酸残基序列,其余检测项目按《中国药典》三部(2005版)规定进行。结果1批rhBMP-2原液鉴定结果显示,样品比活性为5.9×104BU/mg,纯度为97.6%,N-末端序列为(MKR)LKSSCKRHPLYV,其余项目检测结果均符合《中国药典》三部(2005版)的要求。结论该质控标准可用于rhBMP-2产品的常规检定。     

重组人肿瘤坏死因子相关凋亡诱导配体突变体质控方法与质量标准的建立

目的建立重组人肿瘤坏死因子相关凋亡诱导配体突变体的质控方法和质量标准。方法利用乳腺癌细胞MDA-MB-231增殖抑制试验测定重组人肿瘤坏死因子相关凋亡诱导配体突变体的生物学活性;RP-HPLC和SECHPLC测定其纯度;胰酶酶切,HPLC测定其肽图;荧光定量PCR检测E.coli宿主DNA残留量;ELISA法测定宿主蛋白残留量;Edman降解法进行N-末端序列测定;水平等电聚焦电泳法测定等电点;其余检测项目按《中国药典》三部(2010版)规定进行。同时对通常在原液中进行检定的宿主DNA残留、菌体蛋白残留、N-末端序列测定进行初步的方法验证,考察其在成品中进行检测的可行性。结果经初步验证,在成品中进行宿主DNA残留、菌体蛋白残留、N-末端序列测定具有可行性,用建立的方法对重组人肿瘤坏死因子相关凋亡诱导配体突变体成品进行检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》三部(2010版)的要求。结论建立的质控方法和质量标准具有保证产品安全、有效、质量可控的特点,可用于该类产品的常规检定。同时只对成品进行质控的方法和质量标准对同类产品的质量控制具有借鉴意义。     

重组葡激酶-水蛭素融合蛋白质控方法和质量标准的建立

目的建立注射用重组葡激酶(staphylokinase,SAK)-水蛭素(hirudin,HV)融合蛋白(SFH)的质控方法和质量标准。方法采用纤维蛋白平板溶圈(fibrin agarose plate assay,FAPA)法测定SFH的溶栓比活性,纤维蛋白凝块溶解法测定SFH的抗凝比活性;还原型SDS-PAGE测定SFH的相对分子质量;非还原SDS-PAGE和反相高效液相色谱(reversed-phase high-performance liquid chromatography,RP-HPLC)测定SFH的纯度;胰酶裂解后采用RP-HPLC法分析SFH的肽图;其他各项指标的检测按《中国药典》三部(2010版)规定进行。结果用建立的方法对SFH原液和成品进行检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》三部(2010版)的要求。结论建立的质控方法和质量标准能够保证产品安全、有效、质量可控,可用于注射用SFH产品的常规检定。     

聚乙二醇重组人血管内皮抑制素质控方法的建立

目的 建立聚乙二醇重组人血管内皮抑制素的质控方法.方法 采用内皮细胞迁移荧光分析法测定样品的生物学活性;反相高效液相色谱(RP-HPLC)法测定样品的纯度和含量;胰酶消化法测定肽图;其余项目按<中国药典>三部(2005版)和二部进行检测.结果 用建立的生物学活性测定方法测定的3批原液的比活性分别为88.4、56.5和8...     

重组人源抗狂犬病病毒单克隆抗体的质控方法及质量标准的建立

目的建立重组人源抗狂犬病病毒单克隆抗体(rhRMcAb)的质控方法和质量标准。方法采用小鼠中和试验(MNT)和快速荧光灶抑制试验(RFFIT)分别测定6批rhRMcAb样品的中和活性;还原、非还原SDS-PAGE检测rhRMcAb样品的纯度;还原烷基化胰蛋白酶酶切和反相高效液相色谱法分析rhRMcAb的肽图;焦谷氨酸肽酶去除N-端焦谷氨酸封闭后,氨基酸序列分析仪测定rhRMcAb的N-端氨基酸序列;表面等离子共振法(Surface plasmon resonance,SPR)测定rhRMcAb与狂犬病病毒糖蛋白的亲和常数;按《中国药典》三部(2005版)要求检测rhRMcAb的各项其他指标,并建立其质量标准。结果采用MNT和RFFIT2种方法检测6批rhRMcAb的中和活性,批间活性基本一致;3批rhRMcAb原液的还原SDS-PAGE纯度大于99.0%,非还原SDS-PAGE除抗体主带外,在高、低相对分子质量处分别存在一些次带;3批原液样品的肽图图谱与理化测定对照品一致;N-端氨基酸序列与理论序列一致;结合狂犬病病毒糖蛋白抗原的亲和常数为1.86×108/M;其他各项指标均符合《中国药典》三部(2005版)要求;建立的质量标准中,中和活性应不低于500IU/mg,SDS-PAGE及HPLC纯度应不低于98.0%。结论已建立了rhRMcAb的质控方法和质量标准,可用于rhRMcAb产品的检定。     

重组抗血管生成素肽-Fc融合蛋白的质控方法和质量标准的建立

目的建立重组抗血管生成素肽-Fc融合蛋白的质控方法和质量标准。方法分别采用分子筛高效液相色谱(size exclusion-high performance liquid chromatography,SE-HPLC)、反相高效液相色谱(reverse phase-high performance liquid chromatography,RP-HPLC)和还原毛细管电泳(reduced capillary electrophoresis-sodium dodecyl sulfate,rCE-SDS)测定3批重组抗血管生成素肽-Fc融合蛋白成品的纯度;采用AlphaScreen组氨酸检测试剂盒进行重组抗血管生成素肽-Fc融合蛋白受体配体结合试验测定效价;采用ELISA法进行鉴别试验;蛋白含量、pH值、渗透压摩尔浓度、Tween-20、不溶性微粒、外观、澄清度、生物学活性、水分、内毒素、无菌试验、异常毒性、渗透压等项目的检测按《中国药典》三部(2010版)规定进行。结果 SE-HPLC、RP-HPLC及rCE-SDS测定3批供试品的纯度分别为>98%、>80%和>99%;重组抗血管生成素肽-Fc融合蛋白对血管生成素(angiopoietin,Ang)与酪氨酸激酶受体(tyrosine kinase with immunoglobulin-like and EGF-like domains,Tie)结合的抑制作用呈剂量依赖性,3批供试品与标准品的剂量反应曲线相同,呈典型的倒S型,R2均>0.98,结合效价均在平均值的正负25%区间内;3批供试品的S/N值均>1.60;其他项目检测结果均符合《中国药典》三部(2010版)相关规定。结论建立的质控方法具有保证产品安全有效、质量可控的特点,可用于该类产品的常规检定。     

重组人尿激酶原质控方法和质量标准的研究

目的建立重组人尿激酶原的质控方法和质量标准。方法以纤维蛋白平板法测定重组人尿激酶原的生物学活性,S-2444发色底物法测定单双链比例,还原型SDS-PAGE测定相对分子质量,SDS-PAGE和分子筛色谱测定纯度,毛细管电泳法测定等电点,胰蛋白酶酶切后分析肽图,其余检测项目按《中国药典》三部(2005版)规定进行。结果用建立的方法对原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》三部(2005版)的要求。结论所建立的质控方法和质量标准可用于重组人尿激酶原产品的常规检定。     

人源化抗表皮生长因子受体单克隆抗体质控方法的建立

目的建立人源化抗表皮生长因子受体(Epithelial growth factor receptor,EGFR)单克隆抗体的质控方法。方法采用体外细胞生长抑制试验测定人源化抗EGFR单抗的生物学活性;质谱法和还原型SDS-PAGE法测定其准确相对分子质量;去封闭后用Edman降解法测定其N-末端氨基酸序列;SDS-PAGE和分子排阻高效液相色谱(SEC-HPLC)法测定其纯度;反向液相色谱(RP-HPLC)法测定其肽图;定量PCR法和狭缝杂交法测定其外源性DNA残留量;其余项目按《中国药典》三部(2010版)要求进行。结果人源化抗EGFR单抗原液和成品的相对生物学活性分别为(100±20)%和(94±14)%;质谱法测得该单抗参考品轻、重链的相对分子质量分别为23 370.75和50 341.00,相对误差分别为0.005%和0.006%;还原型SDS-PAGE测得该单抗轻、重链的相对分子质量为27 000和53 400;轻、重链N-末端氨基酸序列均与理论一致;单抗原液的还原型SDS-PAGE纯度为98.5%;SEC-HPLC原液纯度为(98.39±0.05)%和成品SEC-HPLC纯度为(98.17±0.04)%;肽图图谱与理化参考品一致;定量PCR测得其外源DNA残留量小于100 pg/剂量;其他各项指标均符合《中国药典》三部(2010版)要求及其他相关要求。结论初步建立了人源化抗表皮生长因子受体单克隆抗体的质控方法,可用于该制品的常规质量控制。     

血管内皮生长因子抑制剂生物学活性检测方法的建立

目的建立血管内皮生长因子抑制剂(VEGF Trap)生物学活性检测方法。方法利用HEK293/D9/Flt-18R(al-pha)/Flt-IL18R(beta),clone V3H9细胞系,通过荧光素酶检测系统(Steady-Glo~ Luciferase Assaysystem)进行VEGF Trap的生物学活性检测,用VEGF Trap参考品计算供试品的相对百分效价,并对该方法进行精密性和准确性验证。结果VEGF Trap供试品及参考品在该方法中均存在量效关系,且符合四参数方程式:y=(A-D)/[1+(X/C)B]+D。3批VEGF Trap原液和6批成品经3次测定,相对百分效价的平均值在(90.00±2.40)%～(116.77±16.50)%之间,变异系数均小于15%。1批VEGF Trap原液及成品经3次测定,回收率分别为(90.40±2.67)%和(117.20±18.12)%。结论已成功建立VEGF Trap生物学活性检测方法,该方法重复性好,准确性高,可作为VEGFTrap生物学活性的常规检测方法。     

狂犬病毒aG株糖蛋白在CHO细胞中的表达

目的克隆狂犬病毒aG株糖蛋白(Glycoprotein)基因,构建重组真核表达载体,在CHO细胞中表达aG株糖蛋白。方法采用RTPCR,从狂犬病毒aG株基因组RNA中扩增GP基因,并将该基因克隆至真核表达载体pCIdhfr上,以脂质体介导法转染CHOdhfr-细胞。用MTX筛选的抗性克隆经ELISA、免疫荧光及Westernblot检测GP蛋白的表达。结果克隆到狂犬病毒aG株糖蛋白全长基因,并在CHO细胞中进行表达。结论成功地在CHO细胞中表达了狂犬病毒aG株的糖蛋白,为进一步开发狂犬病毒基因工程疫苗打下基础。     

Adipose glycerolipid formation: Effect of nutritional and hormonal states

The potential of glycerolipid formation fromsn-glycerol-3-phosphate (GP) and 2-monoacylglycerol (MG) was studied in adipose microsomal fractions under various nutritional and hormonal states. Glycerolipid formation from GP was followed in the presence of [¹⁴C]glycerol-3-phosphate and palmitoyl-CoA and was assayed by measuring the formation of butanol-soluble product, consisting mainly of [¹⁴C]phosphatidate. Glycerolipid formation from MG was determined in the presence of 2-monooleyl glycerol and [¹⁴C]palmitoyl-CoA, and was estimated by the formation of both [¹⁴C]di- and triacylglycerol. Glycerolipid formation from GP was decreased significantly during food deprivation, in experimental diabetes, in the presence of lipolytic hormone, and during aging. Glycerolipid formation from MG did not change under these conditions and continued at the same rate as observed in control animals. The rate of glycerolipid formation from GP was 7–20 times greater than from MG in the various fat depots. Measurement of the adipose mono-acylglycerol concentration did not show any correlation with the glycerolipid formation from MG. The studies suggest that glycerolipid formation from MG is active in various fat depots, and is substantial when glycerolipid formationvia GP is impaired.     

Alumina‐supported microwave synthesis of Cassia marginata seed gum‐graft‐polyacrylamide

In this article, we report the alumina‐supported, microwave (MW)‐induced synthesis of Cassia marginata seed gum‐graft‐polyacrylamide (MWS‐GP). No initiator or catalyst was required in the synthesis, and the conditions for the grafting were optimized by variation of the acrylamide concentration, MW power, and exposure time. At an identical monomer concentration, a higher level of grafting was observed in the solid‐supported method than under aqueous conditions (the MW‐assisted or redox‐initiated thermal method). The used alumina support was easily separated from MWS‐GP and reused for another three cycles without any significant loss in its efficiency as a solid support. MWS‐GP synthesized under optimum conditions was characterized with Fourier transform infrared spectroscopy, ¹³C‐NMR, thermogravimetric analysis, and X‐ray diffraction, with C. marginata gum as a reference. The properties of MWS‐GP and its saponified derivative were studied to explore the applicability areas of the copolymer in hydrogel formation. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Biological evaluation of chitosan‐based in situ‐forming hydrogel with low phase transition temperature

The aims of current study were to choose a method for preparing sterile chitosan‐α,β‐glycerophosphate (CS‐α,β‐GP) in situ‐forming hydrogel which had potential applications in tissue engineering and evaluated its biocompatibility and degradation characteristics. The results of sterilization stability tests indicated that sterile formulations could be obtained by ultraviolet irradiation of CS powders, 0.22 µm filtration of α,β‐GP and lactic acid solutions, and sterile preparation of CS‐α,β‐GP formulations. The obtained sterile CS‐α,β‐GP formulations showed low hemolysis rates and low BSA adsorption at physiological conditions. When injected in vivo the CS‐α,β‐GP sol turned into gel implant in situ and could be degraded gradually. A minimal inflammatory reaction which could not be found by macroscopic evaluation was induced after injection and new capillary formation was found around the hydrogel humps, making the CS‐α,β‐GP hydrogel worthwhile to be considered for tissue engineering and biomedical applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41594.     

高斯过程及其在软测量建模中的应用

结合工业萘初馏塔关键质量指标估计问题，提出了采用高斯过程（GP）建立复杂工业过程软测量方法。将自动相关确定（ARD）原理与GP模型结合进行软测量模型辅助变量选择,通过建立GP软测量模型，同时得到关键质量指标估计值和相应的预测不确定度，有效解决了现有软测量建模方法不能给出估计值的测量不确定度的问题。研究表明，GP软测量模型不仅能自动选择辅助变量，而且还具有较高的估计精度和较小的测量不确定度，能够更好地满足工业现场对测量可靠性的要求。     

Prediction of pressure drop and minimum spouting velocity in draft tube conical spouted beds using genetic programming approach

The smart method of genetic programming (GP) is used to predict the operating pressure drop (ΔP_s) and the minimum spouting velocity u_ms for conical spouted beds (CSBs) equipped with nonporous draft tubes. Accordingly, six dimensionless variables have been taken as model inputs, including crucial parameters associated with the bed and tube geometric and operating conditions. Two general correlations comprising almost all constitutive and operating variables have been derived for the first time by the GP approach. Both ΔP_s and u_ms values predicted by the GP technique are in a fair agreement with the values corresponding to the experiments, with average absolute relative errors (AARE) of 18.9 and 19.9 %, respectively. The results of the proposed correlations show that the GP method is a powerful tool to make reasonable estimates.     

埃博拉病毒包膜蛋白DNA表达质粒对小鼠中和抗体的诱导作用

目的探讨埃博拉病毒(Ebola virus)包膜蛋白GP DNA表达质粒对小鼠中和抗体的诱导作用。方法将质粒pcDNA3.1、pcDNA3.1-GP(全长GP)、pcDNA3.1-GPΔTM(去跨膜段的GP)和pcDNA3.1-GPΔMe(t去起始密码子的GP)分别经肌肉注射免疫BALB/c小鼠,ELISA法检测血清GP抗体滴度;Western blot法检测抗体的特异性;利用埃博拉假病毒感染293E细胞模型观察中和抗体对埃博拉假病毒的中和作用。结果全长的埃博拉病毒GP和去跨膜段的GP可诱导有效的免疫反应,且诱导生成的GP抗体特异性良好;GP抗血清可有效抑制埃博拉假病毒进入293E细胞。结论埃博拉病毒GP DNA表达质粒可诱导高滴度的中和抗体,并可有效中和埃博拉假病毒。     

狂犬病毒aG株核蛋白和糖蛋白双表达质粒的构建及其在真核细胞中的表达

目的构建狂犬病毒aG株核蛋白(NP)和糖蛋白(GP)双表达重组质粒,在中国仓鼠卵巢细胞(CHO-K1)中表达核蛋白和糖蛋白。方法提取狂犬病毒RNA,应用RT-PCR方法扩增NP和GP基因,分别将其克隆到pIRES载体上,获得同时含有NP和GP基因的双顺反子重组质粒pING,并以脂质体介导法转染CHO-K1细胞,G418筛选,用ELISA和IFA检测NP和GP的表达。结果限制性内切酶分析表明重组质粒pING含有NP和GP基因片段,长度分别为1353bp和1575bp。应用ELISA和IFA方法,在转染细胞中均检测到NP和GP的表达。结论重组双表达质粒可在CHO细胞中同时表达NP和GP,为进一步开发重组狂犬病疫苗奠定了基础。     

Elimination of glycidyl palmitate in diolein by treatment with activated bleaching earth

In this study, activated bleaching earth (ABE) was used to eliminate glycidyl esters from both triacyl- and diacylglycerol oils. To investigate the mechanism, glycerol dioleate containing glycidyl palmitate (GP) was treated with ABE and the fate of the GP was monitored by analyzing the feed, treated, and ABE-absorbed oils using a gas-liquid chromatograph equipped with a flame-ionized detector. GP was completely removed from both the treated and absorbed oils. This indicates that this treatment is useful for GE removal from diacylglycerol oil, although it was not achieved by absorption of GE on ABE but rather by modification of GP. The results of composition analysis demonstrate that GP is transformed to glycerol monopalmitate, glycerol palmitate oleate, and glycerol dipalmitate at a recovery rate of 99.1 ± 1.3 %. An increase in glycerol monooleate and trace amounts of free glycerol and fatty acids were also observed after treatment. The transformation is proposed to involve a ring-opening reaction of GP with water contained in the ABE and in the bulk oil followed by an interesterification reaction among the resultant monopalmitate and the glycerol dioleate of the bulk oil. All the generated compounds were simple acylglycerols and glycerol. Therefore, ABE treatment could be useful for GE removal during the manufacture of edible oils.     

Electrolytically exfoliated graphene–polylactide‐based bioplastic with high elastic performance

This work reports an innovative way to prepare biopolymer composite by incorporating graphene (GP) synthesized from electrolytic exfoliation into biodegradable polymer blend (polylactide/epoxidized palm oil: PLA/EPO) based on melt‐blending method and studies their physical properties for food packaging and related applications. Multilayer GP structure synthesized by electrolytic exfoliation is confirmed by transmission electron microscopy and Raman spectroscopy, whereas homogeneous GP incorporation in PLA/EPO is verified by scanning electron microscopy and X‐ray diffraction. From thermogravimetric analysis and heat deformation temperature (HDT) studies, the decomposition and HDTs of PLA/EPO/GP composites are higher than those of PLA/EPO but are lower than those of pristine PLA and tend to decrease with increasing GP content because of thermal conductivity effect. From standard tensile test, loading of GP in PLA/EPO at an optimal concentration of 0.6 wt % results in higher elongation at break by as much as 52%. The observed additional elongation under a given tension and the corresponding lower tensile strength/Young's modulus may be attributed to lower binding force of materials in the composite because of the presence of relatively weak GP–PLA/EPO interfaces. Moreover, oxygen permeability is found to decrease with increasing GP contents and oxygen permeability is reduced by 40.33% at the GP loading concentration of 0.6 wt %. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41439.