抗体药物中双特异性抗体检测方案(液相色谱柱)

TSKgel COLUMNSApplication Note TSKgel COLUMNS SEC/MS Analysis of a Bispecific Antibody Introduction More potent formats of monoclonal antibodies (mAbs)， suchas bispecific antibodies (bsAbs)， are on the rise in the area ofbiotherapeutics. bsAbs recognize two different epitopes. This dualspecificity increases the potency of these molecules compared tomAbs and expands the range of possible applications. bsAbs can beused to redirect T cells to tumor cells， block two different signalingpathways simultaneously，dually target different disease mediators，and deliver payloads to targeted sites. At this time， more than 50 bsAbproducts are currently undergoing clinical evaluation. Characterization of bsAbs is essential to ensuring product safetyand efficacy. Size exclusion chromatography (SEC) coupled withmass spectrometry (MS) is increasingly being used to identify theaccurate molecular mass of biomolecules， including bsAbs. SEC/MS，however， requires the use of mobile phases that do not contain highconcentrations of non-volatile salts and the use of columns that donot exhibit particle shedding which will interfere with the MS signalresponse. In this application note， a bispecific T cell engager (BiTE@) consistingof two single-chain variable fragments (scFvs)recombinantly linked bya nonimmunogenic five-amino-acid chain (Figure 1) was analyzed bySEC/MS using a TSKgelUP-SW3000， 2 pm column. 31Figure 1. Formation of Bispecific T Cell Engager (BiTE) Formation of BiTE Experimental HPLC Conditions Results and Discussion The ~55 kDa BiTE and ~150 kDa parent mAbs were subsequentlyinjected onto a TSKgel UP-SW3000 column coupled to a Q ExactivePlus mass spectrometer for molar mass determination. Figure 2shows the (a) total ion chromatogram， (b) mass spectrum and (c)deconvoluted mass spectrum of the BiTE. A main peak can be seen atm/z 54，143； adjacent peaks at m/z 54，181， 54，219 and 54，086 correspondto different salt adducts. 3FFigure 2. SEC/MS analysis of the BiTE. Accurate molar mass of theBiTE was identified as 54.1 kDa via SEC/MS. Figure 3 shows the (a) total ion chromatogram， (b) mass spectrum and(c) deconvoluted mass spectrum of one of the parent mAbs. A mainpeak can be seen at m/z 149，264； adjacent peaks at m/z 149，426 and149，592 correspond to different glycoforms. Similar results (not shown)were reproduced for the other parent mAb. These results demonstrate accurate molar mass determination for theBiTE and both parent mAbs utilizing a 20 mmol/L ammonium acetate，10 mmol/L ammonium bicarbonate (pH 7.2) mobile phase with SEC/MScompatibility. Figure 3. SEC/MS Analysis of the Parent mAb. Accurate molar mass ofthe parent mAb was identified as 149.3 kDa via SEC/MS. 7.8 100 A 0 A= T 0 2 6 10 12 14 Prior to analysis， a blank injection was run in order to assess columnparticle shedding. Figure 4a shows the total ion chromatogram of ablank injection that was run on a new TSKgel UP-SW3000 column. MSdata indicates that there is no shedding from the TSKgel UP-SW3000column prior to sample injection. Additionally， a blank injection wasrun between each of the sample injections in order to monitor samplecarryover. Figure 4b shows the total ion chromatogram of a blankinjection run between the BiTE and parent mAb. No evidence ofcarryover can be seen in the run after sample injection. The lack ofshedding and carryover indicate that the TSKgel UP-SW3000 column issuitable for use with MS. Figure 4. Column Shedding and Carryover Analysis. No shedding orcarryover was observed via MS total ion chromatogram. Conclusion The TSKgel UP-SW3000， 2 pm SEC column can be used as a platformmethod for bispecific antibody accurate mass determination usingSEC/MS. A MS compatible mobile phase under non-denaturingcondition was successfully used with the TSKgel UP-SW3000 column.No signs of particle shedding or sample carryover， which may interferewith MS signal response， were noted with the TSKgel UP-SW3000column. *SEC/MS analysis was performed by the Wistar Proteomics andMetabolomics Facility (Philadelphia， PA) TOSOH BIOSCIENCE LLC ·Horizon Drive， Suite King of Prussia， PA Tel： · email： info.tbl@tosoh.com·www.tosohbioscience.comAN 双特异性抗体（bsAbs）作为一种新型单克隆抗体，正在生物治疗领域兴起。bsAbs可识别两个不同的抗原表位，这种双重特异性使其比普通的单克隆抗体更具治疗效力，并拓展了其应用范围。双特异性抗体用于将T细胞重新导向肿瘤细胞、同时阻断两个不同的信号通路、针对不同疾病介质的双重锚定，并传递特定药物到目标位点。目前，有超过50个双特异性抗体产品已进入临床评估阶段。对双特异性抗体表征是确保产品安全性及功效的前提。尺寸排阻色谱（SEC）串联质谱（MS）分析法正越来越多的用于确定包括双特异性抗体在内的生物大分子的精确分子量。不过，SEC/MS要求使用不含有高浓度非挥发性盐类的流动相，以及不能使用填料颗粒会脱落的色谱柱，因为这会干扰MS信号响应。本研究采用TSKgel® UP-SW3000、粒径2 μm色谱柱以SEC/MS法对一个双特异性T细胞结合子（BiTE®）进行分析。实验结果表明，该色谱柱可以通过SEC/MS方法对双特异性抗体（bsAbs）的精确分子量进行测定。在TSKgel UP-SW3000色谱柱上可以使用非变形条件下的MS兼容流动相，并且色谱柱不存在填料颗粒脱落或样品残留等现象。