实验设计在重组人生长激素-Fc融合蛋白纯化工艺建立过程中的应用

目的 通过实验设计(Design of Experiment,DOE)对重组人生长激素-Fc(recombinant human growth hormone-Fc,rhGH-Fc)融合蛋白纯化工艺中阴离子交换层析的层析条件进行优化,以降低蛋白原液中宿主蛋白(host cell protein,HCP)含量。方法 利用Minitab 19中DOE功能创建4因子2水平完全析因实验设计,通过DOE对rhGH-Fc纯化工艺中离子交换层析条件(流速、上样量、缓冲液pH、洗脱液盐浓度)进行优化,找出操作空间。结果 建立的DOE模型能准确预测实验结果,其中上样量、缓冲液pH、洗脱盐浓度以及缓冲液pH与洗脱液盐浓度的交互效应对收获液中HCP含量有显著影响。获得的最佳层析条件为：上样量15 mg/mL,流速120 cm/h,洗脱液pH 7.0～8.0,洗脱液盐浓度0.1 mol/L。该层析条件下洗脱液中HCP含量均<400 ng/mg,在确定操作空间的结果范围内满足验证要求。结论 本实验成功应用DOE确定了阴离子交换层析去除HCP的最佳层析条件,为其在生物制药领域的更多应用提供了参考。     

减少重组蛋白TP25在Protein L层析中形成多聚体方法的建立

目的建立减少重组蛋白TP25在Protein L层析中形成多聚体的方法。方法通过在Protein L层析纯化重组蛋白TP25使用的洗脱缓冲液中添加半胱氨酸(L-cysteine,Cys)等保护剂,并采用碱性溶液中和洗脱液的方法优化多聚体去除条件,经非还原SDS-PAGE检测纯化过程中目的蛋白多聚体的变化情况,葡聚糖凝胶Sephadex G25层析去除洗脱液中Cys。结果去除Protein L层析形成多聚体的最适条件为:用含Cys终浓度为100 mmol/L的20 mmol/L柠檬酸溶液进行直接洗脱,并经0. 5 mmol/L的NaOH溶液中和。该条件下纯化的目的蛋白纯度可达95%以上,且多聚体含量大幅减少。葡聚糖凝胶Sephadex G25可有效去除半胱氨酸。结论在洗脱液中添加Cys可有效减少重组蛋白TP25在Protein L层析纯化过程中多聚体的形成。     

精制马抗H5N1禽流感病毒免疫球蛋白疏水层析方法的优化

目的对精制马抗H5N1禽流感病毒免疫球蛋白疏水层析方法进行优化。方法将胃蛋白酶直接加入制备好的高效价马抗H5N1禽流感病毒血清进行消化裂解,再加入饱和度为26%的硫酸铵溶液,盐析沉淀离心一次后,直接进行疏水层析进一步纯化F(ab′)2抗体。结果连续洗脱依次出现6个蛋白峰,50min左右,洗脱峰3开始出现,其中含大部分F(ab′)2抗体,此时硫酸铵摩尔浓度约为1.0～1.1mol/L。阶段梯度洗脱,当硫酸铵摩尔浓度降至1.0mol/L时,大量F(ab′)2片段被洗脱下来,纯度达90%以上。结论优化了精制马抗H5N1禽流感病毒免疫球蛋白的疏水层析方法,降低了硫酸铵用量,减少了离心次数,生产周期也大大缩短。     

新型层析技术在人乳头瘤病毒L1蛋白病毒样颗粒快速纯化中的应用

目的采用新型层析技术快速纯化人乳头瘤病毒(human papillomavirus,HPV)L1蛋白病毒样颗粒(virus-like particles,VLPs)。方法应用新型的基于阴离子交换和分子筛原理的色谱填料CaptoCore 700初纯昆虫细胞表达的HPV L1蛋白VLPs,再用高流速、高分辨力的阴离子交换树脂Capto Q ImpRes进一步纯化。各步纯化的样品经SDSPAGE进行分析,采用ImageQuant图像分析软件分析两步纯化后蛋白的纯度,并对初纯蛋白进行质谱分析;改良Lowry法测定初纯蛋白的浓度,并计算杂蛋白去除率。结果昆虫细胞表达的HPV L1蛋白经CaptoCore 700初纯后,目标蛋白组成的VLPs存在于流穿组分中,经质谱分析,为目的蛋白HPV L1;料液处理量为1个柱体积时,杂蛋白去除率达80%,料液处理量为2个柱体积时,杂蛋白去除率为76%。初纯的蛋白经Capto Q ImpRes进一步层析纯化,HPV L1蛋白的纯度接近100%。结论 CaptoCore 700加Capto Q ImpRes两步层析纯化能够快速高效地纯化HPV L1蛋白VLPs,整个工艺过程简单、快速、高效、易放大,适用于大规模生产。     

复合型阴离子交换层析填料Capto adhere纯化工艺的优化

目的优化复合型阴离子交换层析填料Capto adhere的纯化工艺,并进行验证。方法利用中心复合设计(central composite design,CCD)法对影响复合阴离子交换层析的3个因素(上样液pH值、上样液电导率值、上样量)进行优化,拟合出响应曲面,找出最优参数范围。将优化的Capto adhere层析工艺与亲和层析相结合,纯化CHO细胞表达的IgG1单抗培养液,验证工艺参数的稳定性。结果优化的Capto adhere层析工艺为:上样液pH值6.9,上样液电导值率6.3 mS/cm,上样量300 mg/ml gel;CHO细胞表达的IgG1单抗培养液先经蛋白A亲和层析柱初步纯化,再经优化的Capto adhere层析工艺进一步纯化后,样品纯度达98.9%,蛋白A残留量为0.000 52%,宿主细胞蛋白残留量为0.015%,与预测值比较,结果偏差小于10%,且均符合质控标准,两步层析总收率达86.7%。结论优化了复合型阴离子交换层析填料Capto adhere的纯化工艺,利用亲和层析与Capto adhere复合阴离子交换层析两步法纯化单抗是有效、简便、可行的。     

重组白喉毒素无毒突变体CRM_(197)柱复性及纯化工艺的优化

目的优化重组白喉毒素无毒突变体CRM197(recombinant cross reacting material 197,rCRM197)柱复性及纯化工艺。方法将Sephacryl S-200凝胶柱复性和Q Sepharose FF强阴离子交换层析结合,复性和纯化rCRM197;通过正交试验L9(34)考察凝胶层析复性时流速、离子交换层析纯化的pH值、淋洗液中NaCl浓度和洗脱液中NaCl浓度4种因素对rCRM197纯度及回收率的影响,筛选rCRM197复性及纯化的最佳工艺。结果包涵体经Sephacryl S-200凝胶柱复性后,相对分子质量小于36 000的杂质已被去除;经Q Sepharose FF离子交换层析后,rCRM197纯度达95%以上;在复性时流速为10 ml/min、离子交换层析纯化的pH值为7.5、淋洗液为50 mmol/L Tris-Cl+30 mmol/L NaCl、洗脱液为50 mmol/L Tris-Cl+50 mmol/L NaCl的条件下,rCRM197能达到纯度和回收率的共平衡。结论已筛选出rCRM197复性及纯化的最佳工艺,该工艺操作简便,制备的rCRM197纯度高,适用于rCRM197的产业化。     

A型肉毒抗毒素的层析纯化

目的层析纯化A型肉毒抗毒素,提高免疫球蛋白F(ab′)2的纯度。方法采用阴离子交换层析纯化A型肉毒抗毒素,并通过试验设计(design of experiment,DoE)方法优化缓冲液的pH与电导。结果流穿液中的小分子杂蛋白含量受电导的影响较显著,并随着电导的降低而降低,而pH在试验设计范围内对其影响较小;聚集体/聚合体含量在低pH、高电导时含量较高,高pH、低电导时含量较低;F(ab′)2的纯度主要受电导的影响,并随着电导的升高而降低,在试验设计范围(2~20 ms/cm)内,F(ab′)2的纯度均在90%以上。结论采用阴离子交换层析可有效降低A型肉毒抗毒素中聚集体/聚合体以及部分小分子杂蛋白的含量,提高F(ab′)2的含量。     

重组人睫状神经营养因子的纯化

目的纯化重组人睫状神经营养因子(rhCNTF)。方法破菌液上清经硫酸铵分级沉淀和G-25脱盐后,用QSepharose FF阴离子交换层析初纯,再经Superdex 75 prepgrade凝胶过滤精制,并对纯化产物进行各项检测。结果纯化的rhC-NTF纯度达95%以上,蛋白浓度约为2mg/ml,收率约30%,相对分子质量21600,等电点为6.15,与理论值相符合。Western blot检测证明纯化蛋白能够与特异性抗体结合,并能够明显刺激鸡胚背根神经节突触生长。结论采用盐析、离子交换、凝胶过滤等方法有机组合,成功分离纯化了rhCNTF蛋白。     

离子交换色谱法分离纯化水溶性花菁染料的研究

针对水溶性不对称荧光染料及副产物不同的电荷特性,采用DEAE-52阴离子交换色谱结合葡聚糖凝胶色谱对其进行分离纯化,并以纯化后的染料作为荧光探针对抗体进行标记。结果表明,当使用含0.5 mol/L Na2SO4的0.2 mol/L PBS缓冲液(pH 7.4)进行洗脱时,DEAE-52阴离子交换色谱能够将目标染料分离,得到纯度为98.4%的产物,得率57.1%。产物经活化后用于标记抗体,标记度达到4.6,与同类别的CFTM647相当,达到了实用的要求。     

长效重组人生长激素-Fc融合蛋白纯化工艺的建立

目的建立长效重组人生长激素-Fc(recombiant human growth hormone-Fc,rhGH-Fc)融合蛋白的纯化工艺。方法 rhGH-Fc融合蛋白细胞培养液经离心、低pH处理、过滤、浓缩预处理后,再经Protein-A亲和层析、CM阳离子交换层析、Q HP阴离子交换层析进行纯化,同时检测rhGH-Fc融合蛋白的蛋白含量、纯度、等电点及宿主蛋白、宿主DNA、Protein-A残留量等指标。结果 Protein-A亲和层析、CM阳离子交换层析、Q HP阴离子交换层析纯化产物的蛋白含量分别为145、132、92.4 mg,纯度分别为95. 40%、95. 90%和99. 19%,宿主蛋白残留量分别为2 500、864和71 ng/mg,宿主DNA残留量分别为329、0. 56和0. 12 ng/mg,Protein-A残留量分别为24. 5、3. 2和0 ng/mg。rhGH-Fc融合蛋白相对分子质量约97 300,等电点范围为5. 5~6. 5,可与鼠抗人生长激素(human growth hormone,hGH)抗体发生特异性结合。结论成功建立了rhGH-Fc融合蛋白的纯化工艺,该工艺的纯化产物回收率及纯度均较高,本实验为rhGH-Fc融合蛋白的大规模生产奠定了理论基础。     

Removal of lubricants from effluents from Capron production

Conclusions 1. The composition of water obtained after removing lubricant by the aluminate method and further softening scarcely differs at all from that of softened water and can be re-used in industry.2. The flowsheet described enables a closed purification cycle to be built and finishing waste waters to be used; it enables the water consumption for fibre washing to be reduced, the discharge of toxic caprolactam to reservoirs to be discontinued and caprolactam to be recycled.Kiev Combine. Translated from Khimicheskie Volokna, No. 4, pp. 76–78, July–August, 1969.     

Direct synthesis of LPG from syngas derived from air-POM

Direct synthesis of liquefied petroleum gas (LPG) from a low-cost syngas derived from partial oxidation of methane with air (air-POM) was investigated over a hybrid catalyst consisting of methanol synthesis catalyst and Pd-modified Y-zeolite. The hybrid catalyst demonstrated a high activity and more than 73% selectivity for LPG fraction. BET, NH₃-TPD and TPO-MS were carried out to study the properties of Pd-Y before and after reaction. The results indicated that coke deposition on Pd-Y was the main contribution to the slow deactivation of hybrid catalyst.     

Refractory products from semiacid clays from the Barzassk region

Summary The main advantage of ladle semiacid refractories, having the same life as fireclay, is their low cost owing to the use of cheap, local raw materials and small contents of expensive grog.According to approximate calculations, the saving from the use of local semiacid clays and the reduction in grog consumption should be about 6 rubles per ton of goods at the KMC.     

Zinc-substituted hydroxyapatite produced from calcium precursor derived from eggshells

The availability of natural calcium derived from biowaste materials is an inexpensive alternative to commercial calcium reagents used to produce calcium phosphate bioceramics. This approach is more ecofriendly, cost effective and sustainable owing to its abundant availability. In this study, zinc-substituted hydroxyapatite (ZnHA-Es) was synthesised using calcium precursor derived from eggshells through precipitation method. Zn concentration was varied from 1 mol% to 5 mol% during the synthesis, and the derived powders were calcined at 700 °C in air atmosphere. XRD analysis revealed that ZnHA-Es powders were highly crystalline and comprised biphasic mixtures of HA as the major phase and β-tricalcium phosphate as the minor phase. FTIR examination indicated that all ZnHA-Es samples had a similar structure to pure HA as evidenced by the presence of frequency bands corresponding to phosphate, carbonate and hydroxyl functional groups. The disappearance of vibrational bands of carbonate groups became more apparent with increased Zn concentration. Microstructure analysis revealed that the derived nanostructured particles were spherical (approximately 40–100 nm in diameter) regardless of Zn substitution.