重组抗IgE单克隆抗体天冬氨酸异构化程度分析方法的建立

 目的  建立一种检测重组抗IgE单克隆抗体（单抗）轻链互补决定区中天冬氨酸异构化程度的疏水作用层析（hydrophobic interaction chromatography, HIC）方法。方法  采用木瓜蛋白酶酶切抗IgE单抗，产生抗原结合片段（fragment of antigen binding, Fab）和可结晶片段作为HIC方法的样品。筛选不同品牌及官能团的色谱柱，优化流动相的组成及洗脱梯度，研究样品于5 ℃和﹣20 ℃条件下的稳定性；同时用强制破坏的方式考察该方法的稳定性检测能力。并对该方法的专属性、精密度、线性和准确度进行了验证。结果  采用TSKgel Butyl-NPR色谱柱、流动相甘油含量为5%时色谱图的分离度较好。样品在进样器（5 ℃）中放置24 h或在-20 ℃中放置48 h可保持稳定。该方法能检出强制破坏样品的各成分含量随时间的变化，且专属性良好，重复性及中间精密度均符合规定，Fab段各峰的线性决定系数均≥0.990且准确度均在90%~110%。结论  建立的HIC方法可有效分析单抗制品天冬氨酸异构化程度。     

14型肺炎球菌多糖双抗体夹心ELISA检测方法的建立

目的  建立并验证测定14型肺炎球菌多糖（pneumococcal capsular polysaccharide serotype 14，PS14）浓度的双抗体夹心ELISA。方法  采用Protein G亲和层析纯化兔血清，得到兔抗PS14多克隆抗体（多抗）。以鼠抗PS14单克隆抗体（单抗）为捕获抗体，兔抗PS14多抗为检测抗体，碱性磷酸酶标记山羊抗兔IgG为二抗，PS14为参考品建立双抗体夹心ELISA法。确定鼠抗PS14单抗和兔抗PS14多抗的工作浓度，建立标准曲线，并验证该方法的准确性、精密度和特异性，考察佐剂对该方法的影响。结果  鼠抗PS14单抗的最适工作浓度为5 μg/ml，兔抗PS14多抗的最适工作浓度为1 μg/ml，标准曲线的范围为9.375 ~ 600.000 ng/ml，线性良好，相关系数为0.999。测定多糖样品的PS14回收率为110% ~ 140%，多糖蛋白结合物样品的PS14回收率为90% ~110%。平均批内和批间精密度分别是5.64%和7.14%。13价结合物混合样品的PS14回收率为85% ~100%；含有佐剂的疫苗样品的PS14回收率为85% ~110%。结论  成功建立了双抗体夹心ELISA法，该方法准确性、精密度、特异性良好，且检测结果不受佐剂的影响，具有一定耐用性。     

抗柯萨奇病毒A组2型单克隆抗体制备及ELISA抗原检测方法的建立

目的  制备抗柯萨奇病毒A组2型( coxsackievirus A2,CV-A2)单克隆抗体（单抗）,建立CV-A2抗原检测方法。方法  用纯化后的CV-A2全病毒颗粒免疫小鼠,筛选获得抗CV-A2单抗。建立CV-A2抗原检测方法,确定线性范围,对其准确度、精密度、稳定性、专属性进行验证。用 ELISA检测病毒颗粒纯化过程中样品的抗原含量。结果  制备了高效价的抗CV-A2单抗并建立 ELISA抗原检测方法,检测范围为5.00~320.00 ng/ml。高、中、低3个浓度样品准确度验证回收率在89.58%~104.78%之间。重复性验证变异系数分别为2.10%、2.47%、6.18%。中间精密度验证变异系数分别为2.89%、2.69%、1.94%。耐用性验证回收率在84.26%~114.21%之间。包被微孔板于37℃放置3d,样品回收率在90.31%~103.11%之间。专属性验证结果显示该方法只识别CV-A2抗原,与其他抗原均无交叉反应。结论  建立并验证了CV-A2 ELISA抗原检测方法,可应用于病毒纯化过程中样品的抗原检测,还可应用于含CV-A2的手足口病多价疫苗的CV-A2抗原含量检测。     

全柱成像毛细管等电聚焦电泳分析重组蛋白质药物电荷异质性

目的 分析德谷胰岛素、苏金单抗、阿柏西普和重组人生长激素4种重组蛋白质药物的电荷异质性,为其质量检控提供可靠且快速的分析方法。方法 利用iCE3全柱成像毛细管等电聚焦电泳(imaged capillary isoelectric focusing,iCIEF)分析仪对待测重组蛋白质药物进行检测,将待测样品与载体两性电解质按一定比例混合,在1 500～3 000 V聚焦条件下进行检测。结果 德谷胰岛素等电聚焦电泳图为单峰,等电点平均值为5.25。苏金单抗共检测到4个等电点,其中等电点8.66为主峰,占总峰面积的67.78%。阿柏西普共检测到12个等电点,有6个等电点的峰面积占总峰面积>10%,RSD均<0.10%。重组人生长激素共有3个等电点,其中等电点5.45为主峰占总峰面积的87.60%。结论 利用iCIEF技术能够有效检测德谷胰岛素、苏金单抗、阿柏西普和重组人生长激素的电荷异质性。该方法对4种蛋白质药物的质量检控具有重要意义。     

β-丙内酯残留量检测分析方法的建立及验证

目的  建立准确快速检测灭活疫苗中β-丙内酯（β-propiolactone , BPL）含量的分析方法。方法  采用气相色谱法进行BPL含量检测，用外标法计算BPL含量。对分析方法的专属性、系统适应性、线性和范围、准确度、精密度、检测限、定量限、耐用性进行验证。结果  BPL溶液和供试品溶液在相应保留时间内可见BPL峰，阴性对照溶液未出峰。BPL峰5次进样峰面积相对标准偏差小于2%且与其他峰分离度大于1.5。BPL的浓度在1.11~35.52 μg/ml范围内与峰面积成线性关系。浓度分别为2.22、4.44和8.88 μg/ml的供试品溶液回收率在97.3%~106.9%之间，相对标准偏差均小于3%。检测限为0.30 μg/ml，定量限为0.49 μg/ml。结论  建立的BPL检测方法具有良好的专属性、系统适应性、线性和范围、准确度、精密度、耐用性，符合定量检测的需求。     

A群脑膜炎球菌多糖疫苗中磷含量测定的中国药典方法与欧洲药典方法的比较

目的  比较检测A群脑膜炎球菌多糖疫苗中磷含量的中国药典（2015年版）方法（方法1）与欧洲药典（8.0版）方法（方法2）的差异,为今后的方法选择及进一步优化提供参考。方法  分别用方法1和方法2检测标准磷溶液和A群脑膜炎球菌多糖疫苗中的磷含量,比较这2种方法的标准曲线、定量限、准确度、精密度、耐用性（溶液稳定性）的差异。 结果  方法1和方法2标准曲线的决定系数均值分别为0.997 3和0.999 3,定量限分别为3.97和2.16 μg。检测同一批A群脑膜炎球菌多糖疫苗显示,磷含量检测结果的相对标准偏差,方法1和方法2分别为10.3%和7.6%;磷加样回收率,方法1为87.8%～113.1%,方法2为97.1～103.6%。分别将反应溶液放置0、30、60 min后开始比色,方法1和方法2每30 min吸光度升高均值分别为0.059和0.003。结论  2种方法均可用于A群脑膜炎球菌多糖疫苗中磷含量的检测。与方法1相比,方法2的标准曲线线性更佳、检测定量限更低、准确度和精密度更高、耐用性（溶液稳定性）更好。     

Sabin株脊髓灰质炎灭活疫苗Vero细胞DNA残留定量PCR检测法的验证

目的  验证Sabin株脊髓灰质炎灭活疫苗中Vero细胞DNA残留定量PCR（quantitative PCR,qPCR）检测法。方法  用试剂盒提取Sabin株脊髓灰质炎灭活疫苗原液的DNA,采用qPCR法检测Vero细胞DNA,并验证方法的线性、专属性、准确性、精密度、耐用性及定量限;同时采用DNA探针杂交法检测样品。结果  qPCR检测Vero细胞DNA标准曲线在0.003 0~300.000 pg/μl之间线性良好;对Hep-2细胞、大肠埃希菌、酵母细胞DNA均无扩增反应;高、中、低浓度样品加标回收率在50%~150%,准确性良好;定量检出限为0.003 0 pg/μl;精密度良好（相对标准偏差＜15%）;反应体系配制好后置于2~8 ℃ 30 min后进行检测,相对标准偏差＜15%,耐用性良好。DNA探针杂交法与qPCR结果均小于0.1 pg/μl。 结论  qPCR检测Sabin株脊髓灰质炎灭活疫苗中Vero细胞DNA残留具有良好的线性、专属性、准确性、精密度和耐用性,可适用于该项检测。     

人外周血白细胞处理后CD分子的变化及免疫原性

目的  优化层析-比色法,并对该方法进行分析方法验证;比较优化层析-比色法和层析-积分法,评价两方法测定伤寒Vi精制多糖分子大小的适用性。方法  优化比色法O-乙酰基标准曲线浓度范围,对该方法进行准确度、重复性、中间精密度、线性和耐用性的验证;应用优化层析-比色法和层析-积分法检测多批次伤寒Vi精制多糖分子大小,对结果进行统计学分析。结果 优化层析-比色法准确度回收率均为100%;样品的重复性相对标准偏差（relative standard deviation,RSD）分别为2.4%和0.0%,中间精密度RSD均为3.4%,连续5次测定标准曲线,决定系数均大于0.999 0,耐用性RSD为4.6%。优化层析-比色法和层析-积分法在检测伤寒Vi精制多糖分子大小时,差异无统计学意义（t=-1.372,P＞0.05）。结论  优化的比色法具有良好的准确度、重复性、中间精密度、线性和耐用性,两种方法均可用于检测伤寒Vi精制多糖分子大小。     

疫苗生产洁净车间悬浮粒子的动态监测和趋势分析

目的  对疫苗生产车间半成品配制区域的悬浮粒子进行动态监测和数据分析。方法  采用便携式悬浮粒子采样仪,于2016和2017年对乙型脑炎减毒活疫苗生产洁净车间进行日常悬浮粒子检测,其中A、B级洁净区域为班次检测,C级洁净区域为每周检测。对2年的检测数据制作单值-移动极差控制图,并进行t检验比较。结果  3个级别洁净区域的悬浮粒子数或均值如下,A级区≥0.5 μm的粒子数均<1,≥5.0 μm的粒子数均为0。B级区≥0.5 μm的粒子数均值：2016年为46.82（0～143.21）,2017年为38.83（0～125.33）;≥5.0 μm的粒子数均值：2016年为2.52（0～7.83）,2017年为2.41（0～7.15）。C级区≥0.5 μm的粒子数均值：2016年为214.64（0～679.16）,2017年为209.81（0～619.31）;≥5.0 μm的粒子数均值：2016年为15.04（0～47.13）,2017年为14.16（0～39.24）。3个洁净区域的悬浮粒子数均小于95%置信上限且均符合GMP范围要求。2016和2017年检测数据的差异无统计学意义（t值为0.14～1.02,P值均>0.05）。结论  疫苗生产车间半成品配制区的悬浮粒子动态监测结果符合GMP标准。     

口服脊髓灰质炎减毒活疫苗中庆大霉素残留量检测方法的验证

目的  验证口服脊髓灰质炎减毒活疫苗（人二倍体细胞）中庆大霉素残留量的检测方法。方法  应用庆大霉素ELISA试剂盒建立庆大霉素残留量的检测方法。对该方法标准曲线的线性、线性范围内的准确度、精密度、专属性、耐用性、检测限及定量限进行验证。结果  验证的标准曲线线性良好,相关系数>0.98。该法检测高、中、低浓度的庆大霉素标准品的回收率为92.7%～123.6%;检测同一批次的供试品的相对标准偏差为8%;不同试验人员检测同一批次供试品和不同批次试剂盒检测同一批次供试品,相对标准偏差均<15%;庆大霉素加样回收率为95.9%～121.3%;该法在（37±1） ℃温度范围内耐用性良好;该法测定庆大霉素的检测限为0.040 ng/ml,定量限为0.135 ng/ml。结论  用于检测口服脊髓灰质炎减毒活疫苗（人二倍体细胞）中庆大霉素残留量的方法是有效的。     

Analysis of isoaspartate in a recombinant monoclonal antibody and its charge isoforms

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other proteins, therapeutic mAbs can under go various enzymatic and non-enzymatic reactions that can affect their structural integrity and stability. Among the degradation reactions, isoaspartate (isoAsp) formation is one of the major sources of charge heterogeneity of mAbs. This paper reports the detection and quantification of isoAsp in a recombinant mAb and its charge isoforms resolved by cation exchange high performance liquid chromatography. The assay utilizes the enzyme protein isoaspartyl methyltransferase in conjunction with strong cation exchange separation and UV detection (at 260 nm) of S-adenosyl-L-homocysteine, which is produced stoichiometrically in the enzymatic reaction. The mAb is found to contain an average 0.2 mol of isoAsp per mol of protein, however, various charge isoforms were found to contain different levels of isoAsp. The most acidic isoforms contain approximately 0.7 mol of isoAsp per mol of protein, and no isoAsp is detected in the most basic isoform. It appears that the majority of isoAsp in the mAb is formed as a result of asparagine deamidation.     

重组抗人表皮生长因子受体2单克隆抗体的电荷异质体分析

目的  探讨重组抗人表皮生长因子受体2（human epidermal growth factor receptor 2,Her2）单克隆抗体（单抗）的酸碱异质体对其生物活性和亲和力的影响。方法  分别用过氧化氢、肽-N-糖苷酶F、羧肽酶B对抗Her2单抗进行氧化、脱糖、酶切处理。用阳离子交换高效液相色谱法和毛细管等电聚焦电泳分析抗Her2单抗的电荷异质体,使用微量差示扫描荧光法分析抗Her2单抗的热稳定性,用细胞增殖抑制法和表面等离激元共振技术分析抗Her2单抗的亲和力。结果  脱糖处理抗Her2单抗的酸性电荷异质体增加了5.58%,与免疫球蛋白G Fc受体1（high affinity immunoglobulin gamma Fc receptor Ⅰ,FcGR1A）的亲和力明显减弱,亲和力常数为9.032×10^-8 mol/L。氧化和酶切处理抗Her2单抗的热稳定性降低,去折叠温度分别为63.6和59.5 ℃,均低于未处理抗Her2单抗（66.7 ℃）。结论  3种处理均会使抗Her2单抗产生电荷异质性,脱糖处理抗Her2单抗与FcGR1A的亲和力减弱,氧化和酶切处理抗Her2单抗的热稳定性降低。     

Investigation of Metal-Catalyzed Antibody Carbonylation With an Improved Protein Carbonylation Assay

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.     

FcRn affinity-pharmacokinetic relationship of five human IgG4 antibodies engineered for improved in vitro FcRn binding properties in cynomolgus monkeys

The pH-dependent binding of IgGs to the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. Enhancing interactions between Fc and FcRn via protein engineering has been successfully used as an approach for improving the pharmacokinetics of monoclonal antibodies (mAbs). Although the quantitative translatability of the in vitro FcRn affinity enhancement to an in vivo pharmacokinetic benefit has been supported by several studies, there are also published reports indicating a disconnect in this relation. The body of literature suggests there are likely additional biochemical and biophysical properties of the mAbs along with their FcRn affinity that influence the in vivo pharmacokinetics. Herein, we more broadly evaluate the in vitro Fc-FcRn interactions and biochemical properties of five humanized IgG4 antibodies each with two Fc variant sequences (T250Q/M428L and V308P) and their corresponding pharmacokinetics in cynomolgus monkeys. Our findings indicate that the FcRn affinity-pharmacokinetic relationship does not show a direct correlation either across different IgGs or between the two variant sequences within a platform. Other parameters that have been suggested to contribute to mAb pharmacokinetic properties, such as the pH-dependent dissociation of the FcRn-IgG complexes, mAb biophysical properties, and nonspecific/charge binding characteristics of the mAbs, also did not independently explain the differing pharmacokinetic behaviors. Our results suggest that there is likely not a single in vitro parameter that readily predicts in vivo pharmacokinetics, but that the relative contribution and interplay of several factors along with the FcRn binding affinity are important determinants of mAb pharmacokinetic properties.     

抗重组人骨唾液酸蛋白单克隆抗体的制备及鉴定

目的研制重组人骨唾液酸蛋白(rhBSP)单克隆抗体(mAb),并鉴定其特性。方法以纯化的rhBSP免疫Balb/c小鼠,采用杂交瘤技术制备抗rhBSP mAb;用亚型鉴定试剂条鉴定IgG亚类;ELISA鉴定mAb的特异性和效价。结果获得2株能稳定分泌特异性mAb的抗rhBSP的杂交瘤细胞系AHB1和AHB5,Ig亚类分别为IgG2a和IgG1,轻链均为κ型,其效价分别为1×10-3和1×10-7。腹水mAb经Protein A亲和色谱柱纯化后,纯度达92%以上。结论获得抗rhBSP的mAb,为进一步研究BSP的生物学功能和用于临床诊断实验研究创造了条件。     

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.     

Antibody Responses in Mice to Particles Formed from Adsorption of a Murine Monoclonal Antibody onto Glass Microparticles

Immunogenicity of therapeutic monoclonal antibodies (mAbs) is a concern because of the effects of anti-drug antibodies (ADAs) on therapeutic efficacy. Particulate matter has been suggested as a potential contributing factor to immunogenicity. In this study, we investigated ADA levels in mice in response to administration of a murine immunoglobulin G (IgG)2c/κ mAb (mAb1) that was generated in C57BL/6J mice. Particles of mAb1 were formed by adsorbing the protein to glass microparticles. Formulations containing microparticles were administered subcutaneously to mice of either the syngeneic strain, C57Bl/6J, or the allogeneic strain, BALB/c. ADA levels were measured using an isotype-specific enzyme-linked immunosorbent assay method. Whereas BALB/c mice showed strong IgG1 and IgG2b responses against both the particulate and native mAb1 samples, adsorption of mAb1 to particles rendered it slightly more immunogenic than its native, soluble form. In BALB/c mice, immunoglobulin M (IgM) was produced after the first week of injections and then faded gradually. In contrast, C57BL/6J mice showed moderate IgM, IgG1, IgG2b, and IgG3 responses to injections of glass particle-adsorbed mAb1. ADA responses were higher in the allogeneic BALB/c mice, which do not produce mAbs of the IgG2c/κ isotype. Thus, the presence of both foreign epitopes and particles may be important in inducing ADA responses. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:78–89, 2014     

抗CD19单抗的质量控制研究

目的：研究并建立针对抗白细胞分化抗原19(Cluster of Differentiation 19,CD19)单抗的关键质量属性质控方法。方法：运用肽图对抗CD19的单抗进行鉴别;采用还原/非还原十二烷基磺酸钠毛细管电泳(Capillary Electrophoresis-sodium Dodecyl sulfonate,CE-SDS)和分子排阻色谱(Size ExclusionHigh Performance Liquidchromatography,SEC-HPLC)进行单抗纯度的控制;运用成像毛细管等电聚焦电泳(Imaging Capillary Isoelectricfocusing Electrophoresis,iCIEF)测定电荷异质性;运用高效液相色谱法(High Performance Liquid Chromatography, HPLC)分析抗CD19单抗的糖基化,采用基于报告基因的抗体依赖的细胞介导的细胞毒效应(Antibody-Dependent Cell-mediated Cytotoxicity,ADCC) 测定生物学活性。其他各项指标均应符合现行版《中华人民共和国药典》以及其他相关要求。结果： 肽图检测抗CD19单抗具有相应的特征图谱,能够用于鉴别;还原CE-SDS的轻链与重链峰面积之和百分比为(98.77±0.05)%;非还原CE-SDS主峰峰面积百分比为(96.19±0.05)%,片段百分比为 (3.81±0.05)%;SEC-HPLC单体的峰面积百分比为(99.42±0.001)%,聚合物峰面积百分比为 (0.54±0.001)%;iCIEF分析的主要亚型的峰面积百分比为(66.41±0.38)%,酸性亚型的峰面积百分比为(13.69±0.16)%,碱性亚型的峰面积百分比为(19.90±0.46)%;在糖型分析中,占比最高的三个糖型分别为G0、G0-GN和M5,G0的含量为(64.64±0.61)%,G0-GN的含量为(9.75±0.42)%, M5含量为(6.22±0.35)%,生物学活性的半数最大效应浓度(Concentration for 50% of Maximal Effect, EC₅₀)值为(10.09±1.40)ng·mL^-1。结论：针对抗CD19单抗的关键质量属性,研究并建立了相应的质量控制方法,以确保其安全、有效和质量可控,为该类单抗产品的质量控制方法和策略提供参考依据。