猪脾转移因子生产方法的比较

目的比较从猪脾中提取活性转移因子的不同方法。方法将分别经0 ,3,5次冻融的匀浆液各等分成两份,再分别用Lanrence法和pH调节—超滤法提取转移因子。结果Lanrence法产品不合格;经3次冻融的匀浆液用pH调节—超滤法制备的产品符合国家标准,收率也较高。结论匀浆液冻融次数对转移因子的得率和质量有较大的影响,pH调节—超滤法适合于转移因子的大规模生产     

小牛胸腺肽生产工艺的改进

在生产工艺中提取方法由中性匀浆液冻融提取改为酸性匀浆液提取后.提高了胸腺肽的含量、收率和活性。     

促肝细胞生长素溶液不同制备方法的比较

目的比较从乳猪肝中提取促肝细胞生长素溶液的不同方法。方法对乳猪肝匀浆液分别用反复冻融、酸水解和酶水解3种方法提取，计算单位收率及单位生产成本。结果3种方法制备的促肝细胞生长素溶液均符合国家药品标准，但收率和活性不同。用酸水解法，多肽含量及收率比反复冻融法提高2倍，酶解法多肽含量及收率比反复冻融法提高2．4倍，酸解法和酶解法的活性值比反复冻融法都有所降低。酸水解法的单位成本只有反复冻融法的1／2，酶解法的单位成本与反复冻融法的基本相同。结论3种方法制备的促肝细胞生长素溶液在收率和成本方面有较大的不同，酸水解方法更适合于大规模生产。     

穿琥宁体外稳定性研究

目的:研究穿琥宁的体外酸碱及酶稳定性。方法:将穿琥宁溶解于不同pH值(2.0～9.0)的磷酸缓冲液、人工肠液和不同浓度的肝组织匀浆液中,37℃恒温水浴,于不同时间取样,高效液相色谱法测定穿琥宁的浓度,考察其降解情况或降解动力学。结果:穿琥宁在酸性(pH2.0～5.0)条件下迅速降解(温孵1h后残余量低于10%);在pH7.0磷酸溶液、人工肠液中相对稳定,2h内各取样点的穿琥宁含量均高于95%;37℃下,在50%肝匀浆液中温孵24h后,降解23%;在25%肝匀浆液中温孵24h后,降解15%,且随肝匀浆液浓度的增大而降解加快。结论:穿琥宁在pH<5.0的环境下极不稳定;而胰酶对其几无影响;在肝匀浆液(pH7.0)中不稳定,肝微粒体酶对其有一定影响。     

高效液相色谱法测定颠茄合剂中两组分的含量

目的建立同时测定颠茄合剂中阿托品及羟苯乙酯含量的高效液相色谱法.方法采用CLC-C8色谱柱,流动相为10 mmol·L-1庚烷磺酸钠液(用冰醋酸调pH至3.3)-乙腈-无水乙醇(66286,v/v),流速为1.1mL·min-1,检测波长为210 nm.结果阿托品和羟苯乙酯的线性范围分别为0.5～5.0 mg·L-1(r=0.999 7)、10.0～100.0 mg·L-1(r=0.999 6),平均回收率分别为100.02%、100.24%,RSD不大于1.38%.结论本方法简便、快速、准确,适用于颠茄合剂的质量控制.     

复方奥硝唑栓的制备及质量控制

目的研究复方奥硝唑栓制备工艺及质量控制。方法用高效液相色谱法,20RBA×Eclipse×C8柱（150mm×4.6 mm,5μm）,以甲醇-水-三乙胺（70∶30∶0.3）（含庚烷磺酸钠10 mol·L·mL^-1^-1,用磷酸调pH至4.0）为流动相,检测波长为260 nm,流速为1 mL·min^-1,分别测定奥硝唑、克霉唑的含量。结果奥硝唑在40-240μg·mL^-1,克霉唑在32-192μg·mL^-1线性关系良好,r值分别为0.9998和0.9996,平均回收率分别为99.10%和98.35%。结论复方奥硝唑栓配方合理,制备工艺简单,质控方法可靠,稳定性良好。     

哈蟆油中免疫活性部位的提取工艺与药效学研究

目的:研究哈蟆油中免疫活性部位的最佳提取工艺,并通过药效学实验验证其免疫调节作用。方法:采用正交试验,以酶解液的水解度、收率为指标,利用酶法对哈蟆油中蛋白质进行提取;并通过腹腔注射不同剂量的环磷酰胺复制免疫低下和免疫增强模型小鼠,以脏器指数、吞噬指数为指标,验证哈蟆油中免疫活性部位的免疫调节作用。结果:哈蟆油中蛋白质分2次水解出,第1次水解的最佳工艺为加木瓜蛋白酶2000u·g-1,调pH值至6.0,在45℃下提取4.5h;第2次水解的最佳工艺为固液比为1∶20(V/V),调pH值至6.0,在40℃下提取4.5h;药效学实验证明0.1g·kg-1哈蟆油提取物对免疫低下模型小鼠免疫功能有显著增强作用(P<0.01);0.2g·kg-1哈蟆油提取物对免疫增强模型小鼠免疫功能有显著抑制作用(P<0.01),并对免疫器官有一定的保护作用。结论:所选工艺合理、可行,提取的蛋白质有一定的免疫调节作用。     

咪唑缓冲液对蛋白质测定的影响

咪唑-HCl缓冲液是现代生物化学研究中最广泛使用的缓冲液之一。本文研究了咪哩缓冲液的主要成分对Lowry法测定蛋白质的影响和对鸡胚胎脑匀浆液中蛋白质测定的影响。工作溶液牛血清白蛋白液（BSA）1mg·mL－1用逆渗透水稀释为400，200μg·mL－1。咪叹一HCl缓冲液（pH6．8，50mmol·L-1）含2mmol·L-1MgCl2和40，150，240mmol·L-1KCl；咪哩缓冲液只含40mmol·L-1KCl，不含Mg24。无咪喳缓冲液的标准曲线按传统Lowry法步骤，在IOmL试管中分别加BSAI作液（含蛋白质IO～6Opg）和加水至O．5ml。，然后加3mL新制备的碱性铜试剂（IO…     

猪脾转移因子生产工艺的改进

目的对猪脾转移因子的生产工艺进行改进。方法在原技术基础上,增加了反复冻融工序,于酸性条件下对猪脾转移因子进行提取,比较现行工艺及改进工艺的提取率。结果改进后的生产工艺提高了转移因子的收率(收率由12.4%增加至21.2%)、含量及活性。结论改进后的工艺为利用超滤技术制备高含量制剂提供了基础。     

盐酸赛庚啶片含量测定方法的研究

目的建立盐酸赛庚啶片的反相高效液相色谱含量测定方法,更有效地控制药品质量。方法采用C18柱(150mm×4.6mm,5μm);流动相为乙腈-0.025mol.L-1磷酸盐缓冲液(磷酸二氢钾3.402g,加水至1000mL,内加2mL.L-1三乙胺,并用磷酸调pH值至3.0)35∶65,流速1.0mL·min-1,检测波长为286nm。结果赛庚啶在1～25mg·mL-1范围内线性关系良好,r=0.9997,平均回收率为100.5%(n=9),RSD为0.6%。结论该方法简便,快速,准确。     

Characteristics of Sugar Surfactants in Stabilizing Proteins During Freeze–Thawing and Freeze–Drying

Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein-stabilizing effect during freeze–thawing and freeze-drying. Six enzymes, different in terms of tolerance against inactivation because of freeze–thawing and freeze-drying, were used as model proteins. The enzyme activities that remained after freeze–thawing and freeze-drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze–thawing and freeze-drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze-drying than for the cryoprotection. The comprehensive comparison showed that the C10–C12 esters of sucrose or trehalose were the most effective through the freeze-drying process: the remaining enzyme activities after freeze–thawing and freeze-drying increased at the sugar ester concentrations of 1–10 and 10–100 μΜ, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein-stabilizing effect of a small amount of sugar ester through the freeze–drying process could be enhanced. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1628–1637, 2014     

硫糖铝在体外对抗幽门螺杆菌HpaA IgY的保护作用评价

目的评价硫糖铝对HpaA IgY的保护作用,为制备IgY口服制剂奠定基础。方法大量诱导工程菌DH5α-HpaA-pQE30,制备纯化的重组蛋白HpaA。以HpaA为抗原接种洛曼母鸡,制备纯化的IgY。在不同pH值、含不同浓度胃蛋白酶的HpaA IgY液中加入不同浓度的硫糖铝;将IgY液反复冻融;以ELISA法测定HpaA IgY的剩余抗体活性(AR/AC)。结果在pH 1.5条件下,60%硫糖铝可使IgY抗体剩余活性维持65.8%;在pH 2.0条件下,含30%硫糖铝的IgY可使抗体活性维持85.4%,含50%硫糖铝的IgY可完全维持抗体活性。在pH 3.0条件下,含30%硫糖铝的IgY可使抗体活性维持87.3%,而含40%以上硫糖铝的IgY几乎可完全维持抗体活性。在胃蛋白酶浓度0.02mg/mL条件下,当含10%、30%、50%硫糖铝IgY的剩余活性,在pH值2.0时,分别为62.8%、71.6%、82.5%;在pH值3.0时,分别为75.6%、87.4%、94.5%;反复冻融后,含10%硫糖铝的IgY可使抗体活性提高到70.6%,含30%硫糖铝可使IgY的抗体活性提高到82.7%,50%硫糖铝可提高到89.3%。结论 30%以上的硫糖铝可增强VacA IgY对低pH和胃蛋白酶的耐受能力,抗冻融能力,是较理想的IgY保护剂。     

Investigation of the stabilizing effects of hydroxyethyl cellulose on LDH during freeze drying and freeze thawing cycles

Abstract

The objective of this study was to investigate both the cryoprotective and lyoprotective effects of the polymer hydroxyethyl cellulose (HEC) on the model protein lactate dehydrogenase (LDH) during freeze thawing and freeze drying cycles. The effect of annealing on both protein stability and the physical state of HEC was evaluated. HEC was used as a sole excipient in the protein formulations, and its stabilizing was compared to that of other excipients which are commonly used in freeze dried protein formulations. Furthermore, other quality aspects of the freeze dried samples containing solely HEC were investigated, such as, reconstitution time and product elegance. Protein stability was evaluated functionally by measuring the activity recovery of the model protein LDH. The physical state of HEC after freeze drying was investigated and compared to this of other studied solutes using differential scanning calorimetry and X-ray powder diffractometry. HEC showed superior cryoprotective effects on LDH during freeze thawing, and considerable lyoprotective effects during the freeze drying process. Annealing had limited influence on the stabilizing effect of HEC. The extensive reconstitution times of the HEC lyophilisates could be greatly improved by incorporation of the surfactant Tween 80 into the formulations prior to freeze drying.     

冷冻保护剂对丹参酮脂质体冻融后粒度分布的影响及机理考察

考察了单糖、二糖、三糖、多醇、二甲亚砜以及高分子PVP等附加剂对冻融3次的丹参酮脂质体粒度的影响及机理，结果表明，所选附加剂均能不同程度地抑制丹参酮脂质体在冻融后粒子的显著增大，附加剂对冻融脂质体起保护作用的机理包括脂质体的静电斥力增大和附加剂的空间位阻的协同作用，以及附加剂减少了脂质体周围的冰晶等。     

HPLC法测定酒石酸托特罗定原料含量

目的建立高效液相色谱法测定酒石酸托特罗定原料含量的方法。方法色谱柱为Kromasil C18(200mm×4.6mm,5μm);流动相为甲醇-0.02mol.L-1甲酸铵溶液(用甲酸调pH至3.0)(60∶40),检测波长为283nm,流速为1mL.min-1。结果酒石酸托特罗定在0.0502~0.502mg.mL-1范围内线性关系良好,r=1.000,平均回收率为99.6%,RSD为0.56%(n=9)。结论该方法简便、快速,重现性好。     

Chemical and Physical Stability of Chimeric L6, a Mouse−Human Monoclonal Antibody

Chimeric L6 is a mouse–human monoclonal antibody specific for tumor cell-associated antigens. The factors affecting the physical and chemical stability of chimeric L6 were assessed at elevated temperatures (30–60°C) and by multiple freezing and thawing. Three routes of degradation were observed: chemical degradation to smaller molecular weight species, irreversible aggregation, and formation of a reversible dimer. The specific pathway depended on the stress condition applied and the pH, with maximal overall stability to both thermal stress and multiple freezing/thawing observed at about pH 5.5. Other factors including antibody concentration, buffer concentration, NaCl concentration, and agitation had minimal influence on the stability. Commonly used sugars, polyhydric alcohols, and amino acids effectively prevented freeze/thaw-induced aggregation.     

Impact of Uncontrolled vs Controlled Rate Freeze-Thaw Technologies on Process Performance and Product Quality

Most biomolecules, owing to their marginal stability in liquid state, susceptibility to microbial growth, and tendency to foam upon storage/shipment in the liquid state, often require an alternate method of long-term storage. Cryopreservation is preferred, as it addresses most of these issues associated with liquid storage. However, the stability of the protein in the frozen state depends on the methodology of freezing/thawing and physico-chemical characteristics of the protein. A systematic study was undertaken to understand and evaluate the impact of freezing/thawing method on the process performance and product quality attributes using two freezing methods-conventional freezing in walk-in freezers and thawing in cold rooms using carboys as an uncontrolled rate method, and Celsius/CryoFin? technologies as a controlled rate method. To assess the impact of freeze-thaw cycles on product quality, two types of proteins, a fusion protein and a peptibody (peptide fused to the Fc portion of the antibody), were used, employing appropriate stability-indicating assays. The results demonstrate superior process performance by the controlled rate freeze-thaw technology, both in terms of process times and cryoconcentration, compared to uncontrolled rate freeze thaw technology. Product impact studies indicate that the peptibody is sensitive to the method of freeze-thaw while the fusion protein is not and those that are sensitive to uncontrolled rate freeze-thaw processes can be effectively protected by controlled rate freeze-thaw technologies such as Celsius.     

粉尘螨脂质体的制备及稳定性考察

目的研究冻融法对粉尘螨(Dermatophagoides farinae,DF)脂质体包封率、形态及稳定性影响。方法采用逆向蒸发法制备粉尘螨脂质体悬液,冰冻高速离心法分离脂质体,透射电镜观察脂质体形态。-20℃低温冰箱冰冻,室温融化,反复5次,测定冻融前后脂质体包封率及形态变化,并用离心加速实验测定脂质体稳定性变化。结果经冻融法处理的脂质体包封率明显提高,可达(88.52±0.87)%;在一定范围内包封率随冻融次数的增加而略有增大;经离心加速实验进一步验证了冻融法可使脂质体稳定性提高。结论冻融法为一有效的脂质体制备方法。     

HPLC法测定司帕沙星分散片的含量

目的建立司帕沙星分散片中司帕沙星的高效液相色谱测定方法。方法ODS-C18柱(4.6mm×200mm,5μm);0.05mol.L-1十二烷基硫酸钠溶液(磷酸调pH至3.0)-乙腈(60∶40)为流动相;流速1.0mL.min-1;检测波长275nm。结果司帕沙星浓度在30～70μg.mL-1范围内,与峰面积呈良好的线性关系,r=0.9998,平均回收率为98.8%(n=9)。结论该方法可用于司帕沙星分散片的质量控制。