重组人源性抗HBsAg Fab抗体的肽质量图谱分析

重组人源性抗HBsAg Fab抗体具有较好的特异性和抗原结合活性,为了更好的阐明毕赤 酵母表达的重组人源性抗HBsAg Fab抗体的性质,用基质辅助激光解析飞行时间质谱(MALDI- TOF-MS)对重组Fab抗体的分子质量和肽质量图谱进行了分析。结果显示,毕赤酵母表达的重组 人源性抗HBsAg Fab抗体的分子质量为50678．49Da,与根据其一级结构计算的理论分子质量相 比多2763．84 Da,显示酵母表达的重组Fab抗体为糖蛋白。用胰蛋白酶酶解重组Fab抗体后进行 MALDI-TOF-MS分析显示,大部分的酶解肽段均能检测出来。结果表明毕赤酵母表达的重组Fab 抗体与预期的结构一致。     

在中国发现的一个Hb Q-Thailand(α_2 74(EF3)Asp→His)家系的研究

本文报道了在江西萍乡发现的一例慢速α链异常血红蛋白的研究结果。通过血红蛋白理化性质的测定,异常肽链的柱层析分离,氨乙基化异常肽链用胰蛋白酶酶解后经指纹图谱分析及高压液相层析分离出异常的αT9肽段,一部分进行氨基酸组成测定,一部分直接用DABITC/PITC双偶合法测定整个αT9异常肽段的29个氨基酸的顺序。最后一部分用嗜热菌酶进行第二次酶解,用高压液相层析分离出异常的小片段α70～79,α73～79。同样测定氨基酸组成和用双偶合法进行顺序鉴定。所有的结果均证实本例是α74位门冬氨酸为组氨酸替代,即Hb Q-Thailand(α74[EF_3]Asp→His)。这是中国大陆的首例报道。文中还对血红蛋白的结构分析技术和Hb Q遗传学进行了探讨。     

在中国发现的一个Hb Q-Thailand(α_274(EF3)Asp→His)家系的研究

本文报道了在江西萍乡发现的一例慢速α链异常血红蛋白的研究结果。通过血红蛋白理化性质的测定,异常肽链的柱层析分离,氨乙基化异常肽链用胰蛋白酶酶解后经指纹图谱分析及高压液相层析分离出异常的αT9肽段,一部分进行氨基酸组成测定,一部分直接用DABITC/PITC双偶合法测定整个αT9异常肽段的29个氨基酸的顺序。最后一部分用嗜热菌酶进行第二次酶解,用高压液相层析分离出异常的小片段α70～79,α73～79。同样测定氨基酸组成和用双偶合法进行顺序鉴定。所有的结果均证实本例是α74位门冬氨酸为组氨酸替代,即Hb Q-Thailand(α74[EF_3]Asp→His)。这是中国大陆的首例报道。文中还对血红蛋白的结构分析技术和Hb Q遗传学进行了探讨。     

国内外单抗药物市场概述

单抗药物具有特异性强、不良反应小等优势,近年来在肿瘤和自身免疫性疾病治疗等领域取得了快速发展。目前全球上市抗体药物共55个,2015年销售总额达到916.3亿美元。中国当前正处在抗体药物快速发展阶段,国家食品药品监督管理局(CFDA)共批准生产抗体药物22种,其中国产产品10个,进口产品12个。2014年国内单抗药物市场规模为50.34亿元,随着生物技术的不断发展,国内单抗药物的市场前景将会越来越广泛。     

抗CD52单克隆抗体HPLC-肽图分析方法的建立

建立高效液相色谱(HPLC)-肽图分析方法,用于抗人CD52单克隆抗体的专属性鉴别。抗CD52单抗样品经盐酸胍变性、DTT还原,释放出的游离半胱氨酸残基进行烷基化。超滤置换酶切缓冲液后进行胰蛋白酶酶切并终止。色谱条件:采用Eclipse XDB-C18 4.6×250 mm 5μm(Aglient)色谱柱,0.1%TFA水溶液与0.1%TFA乙腈溶液为流动相,梯度洗脱,检测波长为214 nm,柱温为30℃;质谱条件:分析时长135 min;检测方式正离子,TOF;MS+扫描范围350-1 500 Da;Product Ion+扫描范围100-1 500 Da;质谱分辨率40 000;Exceeds,150 Cps。CD52单抗重链CDR1、CDR3、轻链CDR1对应肽段由质谱鉴定出。HPLC-肽图方法专属性验证显示辅料制剂及异种抗体对检测结果无干扰;精密度验证结果显示目标峰的峰面积RSD%均在1.7%-7.6%之间。且目标峰的保留时间RSD%均在0.1%-0.2%之间,小于5%的可接受标准;耐用性结果显示,3μg胰蛋白酶、37℃和18 h的酶切条件是最合适的样品处理条件。基于CDR相关肽段鉴别的HPLC-肽图分析方法可定性鉴定出抗CD52单抗,方法学验证结果显示该方法适用于抗人CD52单抗的专属性鉴别并可用于质量控制及批检验放行。     

一例Hb Ottawa[αl5(A13)Gly→Arg]的化学结构分析

本文报道在一个8岁汉族女孩血液中发现一种慢速异常血红蛋白。家系调查结果显示,异常基因来自其母。取患者静脉血制备珠蛋白,然后分离异常肽链,进行异常肽链的胰蛋白酶酶解物指纹图谱分析和异常肽段的氨基酸定量分析。结果证明血红蛋白α链第15位甘氨酸被精氨酸所取代,该变异体是Hb Ottawa(α15(A13)G1y→Arg)。本例异常血红蛋白在国内系首次发现。     

LC-MS对奥马珠单抗与其生物类似物CMAB007的分析与结构特征

哮喘是当今世界威胁公共健康的最主要的慢性肺部疾病,作为治疗中重度和难治性哮喘的特效药奥马珠单抗治疗费用相对昂贵,生物类似药CMAB007的研制可以降低治疗费用。本研究用质谱分析一种奥马珠单抗生物类似药CMAB007与原研药的一致性,分别从氨基酸分析、肽图、N/C端序列,还原质谱,寡糖含量,N糖分析等层面对CMAB007和奥马珠单抗进行了系统的比对研究,为进一步临床研究打下基础。结果表明CMAB007和奥马珠单抗氨基酸序列一致,赖氨酸剪切也基本一致,带唾液酸修饰糖形和带核心岩藻糖形比例相近,高甘露糖形比例差异不大,CMAB007略低于奥马珠单抗。因此CMAB007满足生物类似药在结构比对上一致性的要求,有望成为国内首先上市的重组抗人Ig E单克隆抗体药物。     

利用N糖苷酶F对单克隆抗体N糖酶解条件的优化

为了优化利用N糖苷酶F（PNGase F）酶解单克隆抗体中N糖的方法，应用本公司生产的单抗对PNGase F酶的酶解条件进行优化，包括缓冲液pH、酶种类、仪器、酶解程度、变性缓冲液及酶加入量等，总结酶解条件对N糖谱结果的影响。结果显示，置换缓冲液至1×PBS中可以避免某些单抗在低pH酶解时G0F转化为G0F-GN并可改善峰型；快速PNGase F和加入变性缓冲液能有效提高酶解效率；UPLC和1.7 μm粒径色谱柱能提高分离度；不完全酶解影响N糖含量结果。研究结果表明，采用优化的酶解条件可快速、有效的酶解单抗上的N糖，使N糖谱结果准确可靠，为细胞株筛选和单抗药物质量控制提供有效手段。     

rUreB亚单位疫苗中试产品的纯度检测和肽图分析

分析重组幽门螺杆菌尿素酶B亚单位疫苗 (rUreB)中试产品的纯度 ,制备肽指纹图 ,证明rUreB三批中试产品在一级结构上的一致性和生产工艺稳定性。利用面积归一法检测产品的纯度 ,在非还原条件下用TPCK处理过的胰蛋白酶水解 3批中试产品 ,用反相HPLC制备分析肽图。结果显示 ,rUreB的 3批中试产品的纯度达到中试质量要求和肽图分析要求 ,3批中试产品的肽指纹图基本一致 ,重现性好。     

重组人睫状神经营养因子肽图分析

建立重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rhCNTF)的肽图分析方法,用于rh-CNTF的质量控制。胰蛋白酶对rhCNTF进行酶切后,利用RP-HPLC方法对酶切液进行分析,以获得胰蛋白酶切最佳条件及色谱条件,并对连续3批样品进行分析。rhCNTF的胰蛋白酶最佳酶切条件为37℃酶切24h,以A(0.1%TFA-H2O)、B(0.1%TFA-CH3CN)为流动相,采用梯度洗脱的方法对酶切液进行分析,结果连续3批rhCNTF制备产品的肽图完全一致,且其检出峰数目与理论推测值相符。3批产品肽图的一致性为rhCNTF产品的结构同一性提供了有利证据,同时,建立了rhCNTF产品质量控制的一项指标。     

Multispecificity of a recombinant anti‐ras monoclonal antibody

Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m‐ras, a recombinant monoclonal antibody to m‐ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m‐ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m‐ras peptide, it also bound to both recombinant full‐length m‐ras and h‐ras proteins. The cross‐reactive binding of the monoclonal Ab to h‐ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.     

基于抗CD20单克隆抗体的2种夹心ELISA法的建立

目的:建立和比较2种灵敏、特异的夹心ELISA方法,用于准确定量检测食蟹猴血浆中重组抗CD20人源化单克隆抗体(rh-anti-CD20zumab)浓度。方法:以rh-anti-CD20zumab为基础,分别用山羊抗人IgG F(ab')2抗体和驴抗人IgG Fc抗体包被96孔酶标板,加入待测样品,均采用HRP标记的猴血清吸附的山羊抗人IgG抗体进行检测,加底物显色,读取D450nm值。结果:建立了2种定量检测rh-anti-CD20zumab的夹心ELISA方法并进行了确证,样品的前处理分别为1∶20和1∶10,方法的线性范围分别为40~5000和40~12 500 ng/mL,定量下限均为40 ng/mL,两者的板内、板间精密度分别小于16.2%和19.4%,准确度分别为-10.3%~16.6%和-14.4%~12.9%。2种方法均具有良好的特异性和稀释线性,且都未出现钩状效应。结论:方法学确证表明,本研究建立的2种ELISA法均符合新生物制品临床前药代动力学研究指导原则的要求,可用于rh-anti-CD20zumab的检测,为后续rh-anti-CD20zumab在食蟹猴体内的药代动力学研究提供了不同的检测方法。     

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Development and Characterization of Antibodies Against the N Terminus of the Human Dopamine D4 Receptor

Abstract: The human dopamine D4 receptor (hD4R), which has been implicated in human diseases such as schizophrenia and in a personality trait called "novelty seeking," has not yet been characterized at the protein level. Following epitope scanning of the hD4R, we have produced a highly specific monoclonal antibody named DFR1 raised against an amino-terminal peptide in a predicted extracellular region of the receptor. DFR1 decorated recombinant hD4Rs on the surface of intact Chinese hamster ovary (CHO) cells by flow cytometry and fluorescence microscopy and also recognized recombinant hD4.2, hD4.4, and hD4.7 receptor isoforms by western blot analysis. When expressed stably in CHO cells, all three hD4R isoforms contained N-linked glycosylation and showed apparent molecular masses of 48, 55, and 67 kDa for hD4.2, hD4.4, and hD4.7, respectively. DFR1 immunoreactivity representing hD4R protein or dopamine D4 receptor-like antigens was observed in crude membrane extracts of postmortem human brain tissue by immunoblotting. The DFR1 antibody provides a new immunological tool with the potential to further our understanding of the human dopamine D4 receptor protein.     

降钙素原的原核表达及单克隆抗体制备

目的:高效表达与纯化可溶性重组人PCT蛋白,制备高灵敏度和高特异性的抗人PCT医用诊断单克隆抗体。方法:大肠杆菌表达重组人PCT蛋白后,利用饱和硫酸铵沉淀和亲和层析方法纯化PCT蛋白后,经质谱、Western blot和间接ELISA法进行性质鉴定和分析重组蛋白的表达与免疫反应性;重组蛋白免疫小鼠,经细胞融合及筛选制备抗PCT单克隆抗体(m Ab)。结果:在大肠杆菌中高效表达了人PCT蛋白;重组人PCT蛋白具有良好的免疫反应性与免疫原性;经筛选获得7株抗PCT单克隆抗体细胞株,经ELISA鉴定,筛选抗体可与PCT抗原有良好的特异性反应。结论:利用重组人PCT蛋白免疫制备了抗人PCT单克隆抗体,为进一步研发PCT快速诊断试剂提供了原料。     

Antigenic Reactivity of a Monoclonal Antibody Against Amoeba proteus Actin

The reactivity of a monoclonal antibody against actin of Amoeba proteus with actins from other sources was examined. The monoclonal antibody cross-reacted with actins from vertebrate muscles, human erythrocytes, and Acanthamoeba castellanii , but it did not react with Naegleria gruberi actin. The amoeba actin was resolved into 3 bands with isoeletric points of 5.96, 6.03 and 6.10 in electrofocusing gels and they corresponded to 3 peptide spots reacting with the antibody on 2-dimensional immunoblots.     

Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry

A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC–MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC–MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA.     

B3(ds-scFv)靶向超抗原的制备及活性鉴定

To construct the expression vector of a recombinant toxin composed of a disulfide stable single-chain antibody from mAbB3 and SEA(D227A),the binding ability and cytotoxicity of the purified renatured products against the B3 positive carcinoma cells was examined. The VH and VL fragments of the mAbB3 were ligated by overlap PCR, the PCR product was cloned to the pET22b expression vector, then the SEA fragment was inserted into the B3dsscFv-pET22b expression vector which was digested by the same restriction enzymes. The expression plasmid was identified by restriction endonucleases digestion and transformed into E.coli BL21(DE3) followed by IPTG induction. The inclusion body was purified through SP-Sepharose cation exchange column after denaturing and refolding and the binding and cytotoxic ability of the purified products was examined by cell-ELISA and non-radioactive cell proliferation assay seperately. The expression vector B3dsscFv-SEA-pET was constructed successfully and the expression product exists mainly in the inclusion body, amounting to 33% of the total protein. The refolding product remains the binding ability of the single-chain antibody and has cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37℃. This genetically engineered B3dsscFv-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and promises to be an effective reagent for tumor targeted immunotherapy.     

抗HCMV Pp65蛋白单克隆抗体的制备及性质研究

人类巨细胞病毒（Human cytomegalovirus,HCMV）是一种机会性感染疱疹病毒,在人群中感染比较常见,但在免疫缺陷个体与新生儿中可引起严重的疾病。HCMV Pp65是HCMV活动感染的主要标志,也是临床检验检测HCMV感染的重要靶标。为研发抗HCMV Pp65蛋白单克隆抗体作为临床免疫检测HCMV感染的关键原料,本研究采用重组表达的HCMV Pp65蛋白免疫BALB/c小鼠,将免疫小鼠淋巴结细胞与sp2/0细胞融合,采用间接ELISA法筛选阳性克隆与效价测定,再用Western blotting进行抗体特异性鉴定,最后用免疫捕获PCR和免疫荧光法评价其应用前景。最终获得了1株能稳定分泌高效价的抗HCMV Pp65单克隆抗体的杂交瘤细胞株,命名为8D6。Western blotting及间接ELISA检测其效价分别达1∶4 000和1∶105;细胞免疫荧光与免疫捕获PCR实验结果说明,该杂交瘤细胞株分泌的单克隆抗体具有良好的亲和力和特异性,具有作为外周血细胞免疫荧光细胞化学和免疫捕获PCR检测HCMV临床感染的关键原料,也可作为双抗体夹心法检测HCMV临床感染的关键原料,为建立HCMV感染快速灵敏的临床诊断试剂打下了重要的基础。