人用疫苗氢氧化铝佐剂吸附力的质量控制初探

目的 建立氢氧化铝佐剂吸附力的相关质控方法.方法 对3批国产氢氧化铝佐剂与丹麦铝佐剂（Alhydrogel）同时进行粒径、比表面积、零电荷点的测定,并比较两种佐剂对不同浓度牛血清白蛋白（bovine serum albumin,BSA）及百日咳丝状血凝素（filamentous hemagglutinin,FHA）的吸附力.结果 测定显示,3批国产铝佐剂50％体积百分比（DV50）的粒径分布分别为4.49、5.83和6.28 μm;比表面积分别为1 602、1 093和1 014 m^2/kg;零电荷点分别为10.2、10.3和10.2.丹麦铝佐剂DV50的粒径为4.58 μm,比表面积为2 230 m^2/kg,零电荷点为8.5.3批国产和1批丹麦铝佐剂（5 g/L）对2 g/L BSA的吸附率分别为98.69％、99.41％、99.72％和98.04％,均符合欧洲药典的吸附力合格标准.国产和丹麦铝佐剂（1.3 g/L）对50～1 000 mg/L FHA的吸附率均在97％以上.结论 根据对国产氢氧化铝佐剂相关质量指标的研究,初步建立了氢氧化铝佐剂吸附力的质控标准.     

喷雾式添加氨水对氢氧化铝佐剂质量一致性的影响

目的  通过喷雾式添加氨水制备氢氧化铝佐剂,分析其对佐剂质量批间一致性的影响。 方法  分别以喷雾和流加方式添加氨水至三氯化铝溶液各制备3批氢氧化铝佐剂,比较两种氨水添加工艺制备的佐剂粒径、pH、外观等质量指标。 结果  喷雾方式添加氨水制备的3批佐剂平均粒径为（96.7±8.6） nm,pH为4.93±0.02,透析后沉淀量为（20.9±0.4） g/L;流加方式添加氨水制备的3批佐剂平均粒径为（161.0±24.7） nm,pH为4.85±0.06,透析后沉淀量为（105.6±13.8） g/L。相较于流加,以喷雾式添加氨水制备的3批佐剂为均一的乳白色悬液,粒径更小,透析后透析袋底部大颗粒沉淀明显减少,批间一致性更好。 结论  采用喷雾方式将氨水加入三氯化铝溶液中,可使氨水与三氯化铝溶液接触更加充分,产物更加均匀,有助于提高氢氧化铝佐剂外观、粒径、pH等质量指标的批间一致性。     

胞磷胆碱钠注射液中有关物质研究

目的：对胞磷胆碱钠注射液中的有关物质进行分析,考察最大杂质的影响因素.方法：采用XtimateTM C18（250 mm×215;4.6 mm,5 μm）柱,以磷酸盐缓冲液（[0.1 mol·L^-1的磷酸二氢钾溶液和四丁基铵溶液（取0.01 mol·L^-1四丁基氢氧化铵溶液用磷酸调节pH至4.5）等量混合]）-甲醇（95：5）为流动相;流速：1.0 ml·min^-1;柱温：30℃;检测波长：276 nm; 进样量：10 μl.结果：通过47批样品有关物质检查、破坏性试验、异常毒性试验及灭菌温度比较,建立样品的杂质谱,并找出最大杂质的影响因素.结论：该杂质在碱性条件和高温条件下会明显增加,在生产工艺中应控制pH和灭菌温度.     

重组四价人乳头瘤病毒16/18/52/58型病毒样颗粒疫苗制剂工艺的优化

目的 考察不同制剂工艺流程下制备的重组四价人乳头瘤病毒（human papillomavirus,HPV）16/18/52/58型病毒样颗粒疫苗的抗原吸附效果、稳定性及免疫原性,根据实验结果筛选出最合适的制剂配方及工艺。方法  通过改变重组四价HPV疫苗的缓冲体系,以及氢氧化铝佐剂的含量、混合工艺、吸附方式与吸附条件,配制不同的实验疫苗,观察其外观,并分别进行蛋白质稳定性、抗原吸附率和小鼠体内半数有效剂量的检测。 结果 重组四价HPV疫苗原液与450 μg/ml氢氧化铝佐剂在pH6.5的组氨酸缓冲体系下混合后的实验疫苗,外观均一性良好,抗原蛋白稳定性最高,Tm=87.4 ℃,小鼠体内免疫原性较好;搅动吸附条件下的4型别HPV抗原吸附率明显高于静止吸附;铝佐剂和抗原蛋白的吸附与混合先后顺序,不同吸附比例、温度和时间对疫苗外观无显著影响,抗原吸附率均大于99.00%。结论 确定了重组四价HPV16/18/52/58型病毒样颗粒疫苗合适的制剂工艺条件。     

高效液相色谱法测定吲达帕胺片的含量

［摘要］目的建立高效液相色谱（HPLC）法测定吲达帕胺片的含量。方法选用C18柱（4.6 mm×250 mm，5 μm），甲醇 水 冰醋酸（40：60：0.1）为流动相，流速1.0 mL•min 1，检测波长240 nm，柱温为室温，进样量20 μL。结果吲达帕胺在10～80 μg•mL 1浓度范围内与峰面积呈良好的线性关系。回归方程Y＝7.44×107X－5.77×104，r＝0.999 7。平均回收率99.30%（n＝5）。结论该方法定量准确，可靠性强。     

硝苯地平不同粒度对其缓释片剂质量的影响

摘 要 目的：建立硝苯地平原料药粒径测定方法,研究不同粒径硝苯地平原料药对其缓释片（I）体外溶出行为的影响。方法: 采用光散射法对硝苯地平原料药粒径测定进行方法学考察;采用高速万能粉碎机制备不同粒径的硝苯地平原料药样品,用高效液相色谱法测定硝苯地平缓释片（I）的体外溶出曲线,并以国外原研制剂硝苯地平缓释片（商品名：Adalat L,规格：10 mg）为参比制剂,用相似因子f2法进行溶出曲线的相似性比较。结果:粒度测定条件为：粒度分析仪的泵速为1 800 r·min^-1,遮光比为8%～20%,背景与样品的扫描时间为0 s,介质溶液为0.3%吐温80,样品超声时间为1 min。溶出曲线结果表明,硝苯地平原料药粒径越小,溶出越好。Dv90（占总粒子量90%的粒子对应的粒径）从118.781 μm减小至3.471 μm时,自制硝苯地平缓释片在0.25 h时的累积溶出度从11.2%增加至44.0%,溶出曲线与原研制剂溶出曲线相似因子f2先增大后降低,Dv90为29.823 μm时,f2值为77,表明自制片与原研片溶出曲线具有较高的相似度。结论:原料药微粉化技术可显著提高硝苯地平缓释片的体外溶出度,但粒度过细会影响硝苯地平缓释片的缓释效果。为获得与原研品生物等效的制剂,硝苯地平原料药应控制15 μm≤Dv90≤45 μm。     

高效液相色谱法测定还少胶囊中五味子甲素的含量

目的 建立反相高效液相色谱（RP-HPLC）法测定还少胶囊中五味子甲素的含量。方法Diamonsil C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相：乙腈-水-冰醋酸（80：20：0.1），流速为1.0 mL·min^-1,检测波长为254 nm，进样量20 μL,柱温为室温20 ℃。结果五味子甲素在5.7 ～57.0 μg·mL^-1范围内峰面积与浓度具有很好的线性关系，r=0.999 5，平均加样回收率为98.18%，RSD=1.09%（n=5） 。结论该法简便、准确，为还少胶囊的质量控制提供了依据.     

高效液相色谱法测定小儿肠胃康颗粒中芍药苷的含量

［摘要］目的建立测定小儿肠胃康颗粒中芍药苷含量的高效液相色谱（HPLC）法。方法固定相：Alltima C18色谱柱（4.6 mm×250 mm，5 μm）；流动相：乙腈 0.1%磷酸（10.5：89.5）；检测波长：230 nm；流速：1 mL• min 1；柱温：室温；进样量：10 μL。结果芍药苷在0.023 3～1.747 5 μg范围内线性关系良好（r＝0.999 4），平均回收率为100.38%（RSD＝2.20%）。结论该方法简便、准确，可有效控制制剂的质量。     

水质对电解质钙、镁、磷检测结果的影响

目的 探讨两种不同电导率的水质对钙、镁、磷检测结果稳定性方面的影响.方法 选择电导率分别为〈1.0 μS/cm、1.0～2.0 μS/cm和〉3.0 μS/cm水机制备纯水.将入选的75名健康医护人员分成A、B、C 3组,每组随机分配25名.A组水质电导率为〈1.0 μS/cm,B组水质电导率为1.0～2.0 μS/cm,C组水质电导率为〉3.0 μS/cm.将A、B、C 3组人群连续3 d同时采集其血液标本进行钙、镁、磷项目的 检验.最后将3 d的结果进行统计分析.结果 A组在定标当天分别测定的钙、镁、磷结果与定标后第2天和第3天的检测结果间差异均无统计学意义（P 〉 0.05）,并且A组3 d结果均在参考范围内,B组在定标当天分别测定的钙、镁、磷结果与定标后第2天和第3天的结果与定标当天的结果间差异均无统计学意义（P 〉 0.05）,但第2、3天钙、镁、磷有结果超出参考范围上限,C组在定标当天分别测定的钙、镁、磷结果与定标后第2天和第3天的结果与定标当天的结果间差异有统计学意义（P 〈 0.05）,第2、3天钙、镁、磷有结果超出参考范围上限.结论 不同电导率的水质影响钙、镁、磷检测结果,电导率〈1.0 μS/cm的钙、镁、磷检测结果稳定性较电导率1.0～2.0 μS/cm的钙、镁、磷检测结果稳定性好,而电导率〉3.0 μS/cm的钙、镁、磷检测结果稳定性较差.     

高效液相色谱法测定足宁颗粒中盐酸小檗碱含量

目的建立测定足宁颗粒中盐酸小檗碱含量的高效液相色谱法。方法采用高效液相色谱法,用Dionex C18色谱柱（4.6 mm×250 mm,5 μm）,以乙腈 0.1%磷酸（50：50）为流动相,流速为1.0 mL•min－1,检测波长265 nm,柱温为室温。结果盐酸小檗碱在5～50 μg范围内线性关系良好（r＝0.999 9,n＝6）,平均回收率100.201%,RSD＝0.49%。结论该法简便、准确、重复性好,可作为足宁颗粒产品质量的定量依据之一。     

Effect of Thermal Treatment During the Preparation of Aluminum Hydroxide Adjuvant on the Protein Adsorption Capacity During Aging

Six aluminum hydroxide adjuvants, poorly crystalline aluminum oxyhydroxide (AlOOH) were prepared using different thermal treatments of amorphous aluminum hydroxide (Al(OH)₃) in an effort to increase the protein adsorption capacity. All of the adjuvants initially exhibited a higher protein adsorption capacity. However, the protein adsorption capacity decreased during aging at room temperature. X-ray and differential centrifugal sedimentation analysis revealed that complete dehydration of amorphous aluminum hydroxide to aluminum oxyhydroxide is required to produce a stable adjuvant. Any residual amorphous aluminum hydroxide will spontaneously transform to crystalline aluminum hydroxide during aging at room temperature. Since crystalline aluminum hydroxide has a small surface area, the protein adsorption capacity of adjuvants containing amorphous aluminum hydroxide decreased by 30–40% when stored for 6 months at room temperature.     

Effect of thermal treatment during the preparation of aluminum hydroxide adjuvant on the protein adsorption capacity during aging

Six aluminum hydroxide adjuvants, poorly crystalline aluminum oxyhydroxide (AlOOH) were prepared using different thermal treatments of amorphous aluminum hydroxide (Al(OH)3) in an effort to increase the protein adsorption capacity. All of the adjuvants initially exhibited a higher protein adsorption capacity. However, the protein adsorption capacity decreased during aging at room temperature. X-ray and differential centrifugal sedimentation analysis revealed that complete dehydration of amorphous aluminum hydroxide to aluminum oxyhydroxide is required to produce a stable adjuvant. Any residual amorphous aluminum hydroxide will spontaneously transform to crystalline aluminum hydroxide during aging at room temperature. Since crystalline aluminum hydroxide has a small surface area, the protein adsorption capacity of adjuvants containing amorphous aluminum hydroxide decreased by 30-40% when stored for 6 months at room temperature.     

Effect of the strength of adsorption of HIV 1 SF162dV2gp140 to aluminum-containing adjuvants on the immune response

The importance of the strength of antigen adsorption by aluminum-containing adjuvants on immunopotentiation was studied using HIV 1 SF162dV2gp140 (gp140), a potential HIV/AIDS antigen. The strengths of adsorption by aluminum hydroxide (AH) adjuvant and aluminum phosphate adjuvant, as measured by the Langmuir adsorptive coefficient, were 1900 and 400 mL/mg, respectively. The strength of adsorption by AH was modified by pretreatment of AH with two different concentrations of potassium dihydrogen phosphate to produce phosphate-treated aluminum hydroxide adjuvants having adsorptive coefficients of 1200 and 800 mL/mg. The four adjuvants were used to prepare vaccines containing either 1 or 10 μg of gp140 per dose. Antibody studies in mice revealed that the presence of an adjuvant increased the immune response in comparison with a solution of gp140 when the dose was 1 μg. Furthermore, the immune response was inversely related to the adsorptive coefficient. In contrast, no significant difference in immunopotentiation was observed between treatments in the presence or absence of an adjuvant when the dose of gp140 was 10 μg. Analysis of the binding of gp140 to CD4 and anti-gp140 monoclonal antibodies by surface plasmon resonance suggests that tight binding induced structural changes in the antigen.     

Effect of the degree of phosphate substitution in aluminum hydroxide adjuvant on the adsorption of phosphorylated proteins

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.     

Aluminum compounds as vaccine adjuvants

Aluminum compounds are the only adjuvants used widely with routine human vaccines and are the most common adjuvants in veterinary vaccines also. Though there has been a search for alternate adjuvants, aluminum adjuvants will continue to be used for many years due to their good track record of safety, low cost and adjuvanticity with a variety of antigens. For infections that can be prevented by induction of serum antibodies, aluminum adjuvants formulated under optimal conditions are the adjuvants of choice. It is important to select carefully the type of aluminum adjuvant and optimize the conditions of adsorption for every antigen since this process is dependent upon the physico-chemical characteristics of both the antigens and aluminum adjuvants. Adsorption of antigens onto aluminum compounds depends heavily on electrostatic forces between adjuvant and antigen. Two commonly used aluminum adjuvants, aluminum hydroxide and aluminum phosphate have opposite charge at a neutral pH. The mechanism of adjuvanticity of aluminum compounds includes formation of a depot; efficient uptake of aluminum adsorbed antigen particles by antigen presenting cells due their particulate nature and optimal size (<10 μm); and stimulation of immune competent cells of the body through activation of complement, induction of eosinophilia and activation of macrophages. Limitations of aluminum adjuvants include local reactions, augmentation of IgE antibody responses, ineffectiveness for some antigens and inability to augment cell-mediated immune responses, especially cytotoxic T-cell responses.     

Association between immunogenicity and adsorption of a recombinant Streptococcus pneumoniae vaccine antigen by an aluminum adjuvant

The impact on immunogenicity of the degree of adsorption of three Streptococcus pneumoniae (Sp) vaccine antigens to aluminum adjuvants was studied. The three antigens evaluated (Sp1, Sp2 and Sp3) were highly adsorbed by aluminum hydroxide adjuvant, but not adsorbed by aluminum phosphate adjuvant. All of the Sp antigens adjuvanted with aluminum hydroxide elicited higher antibody responses in mice than formulations prepared with aluminum phosphate or non-adjuvanted antigen. Varying the percent aluminum-bound Sp antigen in the formulated vaccine affected the observed antibody responses. These observations suggest that the antibody response observed for Sp antigens in this study is stimulated by a depot effect of the antigen bound to an aluminum adjuvant.     

The Importance of Surface Charge in the Optimization of Antigen–Adjuvant Interactions

The adsorptive behavior of the recombinant malarial antigens R32tet32, R32NS181 and NS181V20 to aluminum hydroxide and aluminum phosphate gels was studied as a function of pH and buffer ions. The Plasmodium falciparum antigen, R32NS181, and the P. vivax antigen, NS181V20, with isoelectric points (pI) of 5.9 and 5.5, respectively, adsorbed readily to the positively charged boehmite form of aluminum hydroxide gel. These two antigens displayed reversible, linear adsorption behavior in the pH range 5–9, with maximal adsorption observed at the lowest pH studied. The addition of acetate buffer ions had little effect on adsorption, while the presence of phosphate decreased adsorption for R32NS181 and NS181V20 by 25 and 40% respectively. The adsorptive behavior of these two antigens with the negatively charged adjuvant, aluminum phosphate, was markedly decreased. The converse situation was observed with the R32tet32 antigen, whose pI is estimated to be 12.8. There was minimal interaction of this antigen with aluminum hydroxide gel except in the presence of phosphate counter ions and significant, nonreversible adsorption with aluminum phosphate gel. Enhanced adsorption of R32tet32 to aluminum hydroxide gel in the presence of phosphate is suggested to be the result of a covalent bond between a surface aluminum and a phosphate anion that modifies the surface charge of the aluminum hydroxide gel. These results indicate that the role of complementary surface charges, both for the ionization state of the protein and for the aluminum adjuvants, is the key in optimizing conditions for significant antigen-adjuvant interactions.     

Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)Al MAS NMR

The present study explores the use of (27)Al magic-angle-spinning (MAS) NMR for the characterization of aluminum hydroxyphosphate adjuvants. Adjuvants were prepared by two different methods: batch-precipitation and precipitation at constant pH, using a wide range of different conditions. The adjuvant compositions showed no evident stoichiometric restrictions and varied as a function of the precipitation conditions. All the aluminum hydroxyphosphate adjuvants were found by (27)Al MAS NMR to contain both tetrahedrally and octahedrally coordinated aluminum. The octahedral form was always predominant. The chemical shifts corresponding to octahedral aluminum were at values intermediate between that of aluminum hydroxide (9 ppm) and those of phosphate-containing aluminum minerals such as variscite (-9 ppm) and varied with the phosphate content of the adjuvant. This was true even for adjuvants precipitated above pH 6 indicating that the phosphate is incorporated into the bulk solid phase contrary to predictions in the literature. Aside from the presence of tetrahedral and octahedral aluminum, there was no evidence in any of the adjuvants of distinct, structurally defined phases indicating that the adjuvants are not mixtures of distinct phases which differ significantly in the number of phosphorus atoms in the next-nearest-neighbor (NNN) position to aluminum.     

Measuring the surface area of aluminum hydroxide adjuvant

The traditional method of determining surface area, nitrogen gas sorption, requires complete drying of the sample prior to analysis. This technique is not suitable for aluminum hydroxide adjuvant because it is composed of submicron, fibrous particles that agglomerate irreversibly upon complete removal of water. In this study, the surface area of a commercial aluminum hydroxide adjuvant was determined by a gravimetric/FTIR method that measures the water adsorption capacity. This technique does not require complete drying of the adjuvant. Five replicate determinations gave a mean surface area of 514 m(2)/g and a 95% confidence interval of 36 m(2)/g for a commercial aluminum hydroxide adjuvant. The X-ray diffraction pattern and the Scherrer equation were used to calculate the dimensions of the primary crystallites. The average calculated dimensions were 4.5 x 2.2 x 10 nm. Based on these dimensions, the mean calculated surface area of the commercial aluminum hydroxide adjuvant was 509 m(2)/g, and the 95% confidential interval was 30 m(2)/g. The close agreement between the two surface area values indicates that either method may be used to determine the surface area of aluminum hydroxide adjuvant. The high surface area, which was determined by two methods, is an important property of aluminum hydroxide adjuvants, and is the basis for the intrinsically high protein adsorption capacity.