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利妥昔单抗生物类似药国内外研发及申报概况
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利妥昔单抗生物类似药国内外研发及申报概况 上海迈泰君奥生物 每日单抗 还记得2015年感动了无数观众的电影《滚蛋吧！肿瘤君》么？这部震撼心灵的影片带给我们的不仅仅是感动和欢笑，还有生命背后的深思...
感动众多网友的漫画家熊顿、著名《新闻联播》主持人罗京、前中国国奥主力球员张亚林、正值青春年华的演员李钰、徐婷……夺走这些鲜活生命的元凶正是近年来全球增长最迅速的恶性肿瘤之一——淋巴瘤。据统计，全世界每9分钟就有1个淋巴瘤新发病例，我国每年死亡病例超过2万人。
那么，得了淋巴瘤就等于宣布死亡了么？ 当然不会，在科技如此发达的当今社会，只要确诊初期用对方案，淋巴瘤患者5年存活率可达50%以上。 对于淋巴瘤患者，化疗为经典的治疗方式，然而传统化疗带来的毒副作用，常让不少患者忧心忡忡。在科技日新月异的当下，抗体药物的快速发展为癌症患者带来了福音。由于抗体具有高特异性、靶向专一性等特点，结合抗体药物的治疗方案能够明显降低毒副作用，减轻患者的痛苦。今天，小编就为大家详细讲解淋巴瘤的天敌——利妥昔单抗~ 利妥昔单抗（Rituximab）由罗氏公司于1997年在美国申请获得上市全球最畅销的单抗类药物之一用于复发难治性滤泡性淋巴瘤的治疗等美国商品名为Rituxan，欧洲商品名：MabThera，中国的商品名：美罗华 国内外销售情况利妥昔单抗业绩斐然，2014年全球销售额75亿美元；2015年73亿美元；2016年上半年38亿美元。但FiercePharma近期预测在2020年美罗华业绩将下滑至50亿美元（原因推测：由于生物类似药的推进较快，申报单位多，导致原研药的市场份额被挤占）。 国内2000年批准利妥昔单抗进口，在2015年PDB国内样本医院中，美罗华销售额统计为15.9亿人民币。
那如此畅销的单抗药物，必然引得各制药公司竞相研发，抢占市场先机，下面来看看目前国内外Rituximab研发及申报情况吧~
国内申报情况 已申报生产及已批准进行临床（含不批准）大约13家：
新药临床试验申报往往需要经过严格的审批程序，那么对于Rituximab生物类似药如何顺利通过申报？有哪些策略和建议值得参考和借鉴？下面大批纯干货来袭啦~1国内以biosimilar申报策略进行临床试验情况
以Biosimilar申报策略进行临床试验的要求 PK，PD，PK/PD，Efficacy有效性，Safety安全性，Immunogenicity免疫原性，Labeling （Extrapolation of Indications）标签（外推适应症）
借鉴Rituximab原研药的临床信息 参考在中国上市的美罗华的说明书内容：·
非霍奇金淋巴瘤·
298例接受单剂或多剂利妥昔单抗、单药或与CHOP联合治疗的NHL患者的群体药代动力学分析结果显示，非特异性清除率（CL1）、可能受B细胞或肿瘤负荷影响的特异性清除率（CL2）以及中央室分布容积（V1）的典型人群估计值分别为0.14L/day、0.59L/day和2.7L。利妥昔单抗的中位终末消除半衰期估计值为22天（范围6.1天至52天）·
161例接受375mg/㎡静脉输注4周的患者数据显示，患者基线CD19阳性细胞计数和可测量肿瘤病灶大小会一定程度影响利妥昔单抗的CL2变化。CD19阳性细胞计数高或肿瘤病灶多的患者CL2较高。但是，在对CD-19阳性细胞计数和肿瘤病灶大小进行校正后，CL2的个体差异仍然较大。V1值随体表面积（BSA）和CHOP治疗方案发生变化。受BSA范围（1.53至2.32㎡）以及伴随的CHOP治疗方案影响的V1变异（27.1%和19.0%）相对较小。·
年龄、性别、种族和WHO体能状况对利妥昔单抗的药代动力学参数没有影响。该分析结果显示按照任一检验协变量调整利妥昔单抗剂量并未明显减低其药代动力学变异性。·
203例首次接收利妥昔单抗治疗的NHL患者，接受利妥昔单抗的375mg/㎡每周静脉输注给药，连续4周。第4次输注后的平均Cmax为486μg/ml（范围77.5-996.6μg/ml）利妥昔单抗的峰谷血清水平与血液CD-19阳性B细胞计数和肿瘤负荷基线值负相关。和无缓解者相比，缓解患者的中位稳定状态血清水平相对较高。国际的工作分类法(IWF)的B、C和D亚型患者的血清水平高于A亚型者。·
在完成末次治疗后3-6个月时，仍可在患者血清中检测到利妥昔单抗。·
37例NHL患者接受利妥昔单抗375mg/㎡每周静脉输注，共8周。平均Cmax值随着利妥昔单抗连续输注而增加，平均Cmax值从首次输注利妥昔单抗后的平均243μg/ml（范围：16-582μg/ml）上升到第八周的550μg/ml（范围：171-1177μg/ml）。·
6次375mg/㎡利妥昔单抗联合6个疗程CHOP方案化疗，利妥昔单抗药代动力学特征与利妥昔单抗单药相似。 Rituximab在中国以Biosimilar申报策略进行临床试验的设计建议
PK试验设计建议：·
由于半衰期较长，建议平行对照设计：T1/2:22 days （6-52days）·
目标人群：建议选择CD20阳性的NHL患者，由于特异性消除受肿瘤负荷大小的影响，因此选择CR患者。·
Dosage：375mg/㎡，用药途经：IV 。·
半衰期22天，而由于每周用药的累积效应，会抹平或者放大试验用药与对照药之间的差异，因此建议单次用药。·
主要终点指标：建议AUC。而Cmax等可作为次要终点指标。·
等效性范围：80-125%。·
试验样本量：至少不能低于18-24对备注：临床试验的参比试剂不能使用biosimilar产品，推荐使用原研药企业在中国销售的同一个产地的产品。海外的Rituximab临床PK设计举例：·
FL（Merck）：n=22，Primary endpoint：safety，sencondy endpoint：Cmax，375mg/㎡IV，+CVP，Single arm·
RA（MERCK）：n=180，parallel design，AUC0-84d，Cmax·
RA（Pfizer）：n=210，Three arms（SBP/USRP/EURP），AUC、Cmax。-PD data-CD19+B-cell count and circulating IgM；change in DAS28，the C-reactive protein and patient’s global assessment of disease activity.-Clinic Response-disease activity score；ACR20,50,70 response- Detection of anti-drug antibodies measured by specific assay.·
RA（Sandoz）：n=164，PK/PD, Efficacy，Safety·
RA（Celltron）：n=147，+MTX, Cmax 1000mg·
RA（Celltron）：n=361（Phase I/III），AUC，Cmax，Efficacy，Safety，1000mg·
FL（Celltron）：n=250(Phase I/III), AUCtau and C EFFICACY, Safety 375mg/㎡ IV PD指标：： ·
由于用药后出现B细胞活性下降，但是该指标与临床疗效不相关。鉴于没有很好地PD指标可提供进行临床评价，因此在临床试验中必须重新选择临床疗效指标。
基于biosimilar申报策略临床比对试验设计建议 ·
临床适应症及治疗方案：鉴于在中国美罗华的获批的适应症仅为非霍奇金淋巴瘤（NHL），因此基于Biosimilar申报策略的临床比对试验的适应症只能做NHL。在充分考虑了原研药临床试验情况后，得知单药更敏感，而与CHOP联用不影响PK 曲线 ,同时由于弥漫性大B细胞非霍奇金淋巴瘤的发病率高于滤泡型非霍奇金淋巴瘤，所以可设计为针对CD20阳性的弥漫性大B细胞非霍奇金淋巴瘤（DLBCL）病人，利妥昔单抗+CHOP治疗方案，与原研药进行头对头比对试验。·
剂量: 375mg/㎡， 给药途径：IV·
主要终点指标：ORR；·
等效性界值Margin：12%·
样本量：企业根据试验设定、终点指标、等效性界值和检验效能等参数及自身情况综合考虑。附 NHL适应症设计参考表
2国内以biosimilar申报策略进行临床试验情况 复宏汉霖 2014年3月,复宏汉霖HLX01（重组人鼠嵌合抗CD20单克隆抗体注射液）获得CFDA非霍奇金淋巴瘤(NHL)的Ⅰ、Ⅱ、Ⅲ 期临床试验批件，2015年3月该适应症开启Ⅲ期临床。目前三期进行顺利。试验详情：2014年开展了随机化、双盲、平行对照比较HLX01与美罗华在CR/CRu CD20阳性的B细胞淋巴瘤受试者中的药代动力学和药效动力学，并评估安全性的 Ib期临床研究，招募病例数80人。试验组为HLX01，静脉注射，375mg/m2，单次给药；对照组为美罗华，静脉注射，375mg/m2，单次给药。主要终点指标：自给药起3个月，PK分析比较HLX01和利妥昔单抗注射液的AUC0-∞。 点评：设计较为合理。
2016年9月，治疗弥漫性大B细胞淋巴瘤相关临床试验患者招募将结束，研究内容为多中心、随机、双盲、阳性平行对照比较重组人鼠嵌合抗CD20+单克隆抗体（HLX01）联合CHOP方案（H-CHOP）与利妥昔单抗（MabThera）联合CHOP方案（R-CHOP）在弥漫性大B细胞淋巴瘤（DLBCL）初治患者中的III 期临床疗效及安全性的临床研究。招募病例人数为320人。试验组为H-CHOP，试验用药HLX01，静脉注射，剂量：375mg/m2；对照组为R-CHOP，对照用药美罗华，静脉注射，剂量：375mg/m2；单次给药。主要终点指标：自给药起21周，总缓解率（ORR）。 点评：设计较为合理，样本量偏小。 信达生物 信达生物的抗CD20单抗，DLBCL适应症与美罗华头对头的III 期临床也于2016年8月启动，据悉信达此项临床将花费上亿，招募病例人数为400例。试验详情：2016年开展了IBI301在经治疗达客观缓解的B细胞非霍奇金淋巴瘤患者中进行的开放、剂量递增Ia期研究，为单臂、非随机化、开放性试验，招募人数9-18人。试验用药：注射剂，剂量分别为125mg/m2（低剂量组）、375mg/m2（中剂量组）和500mg/m2（高剂量组），静脉输注,单次给药。主要终点指标：给药后85天，评价IBI301在经治疗达客观缓解的B细胞淋巴瘤患者中单次给药的耐受性和安全性。 点评：若按照biosimilar申报策略进行该试验，可不进行剂量摸索；需进行与原研药的比对试验，样本量至少18-24对。该试验设计似乎为以新药申报策略进行的I期临床。
2016年8月开始招募随机、双盲、平行对照的针对DLBCL适应症的重组人-鼠嵌合抗CD20单克隆抗体注射液IBI301联合CHOP方案（I-CHOP)与利妥昔单抗联合CHOP方案（R-CHOP）比较的III期临床研究。招募CD20阳性DLBCL患者400例。试验用药I-CHOP： IBI301注射液，静脉注射（IV），一天一次，每次375mg/m2，在第1天给药，每21天一个治疗周期，连续给药共计6个周期；R-CHOP剂量：利妥昔单抗注射液，静脉注射（IV），一天一次，每次375mg/m2，在第1天给药，每21天一个治疗周期，连续给药共计6个周期。主要终点指标：18周的总缓解率。 点评：试验设计较符合biosimilar申报策略的临床试验要求。海正药业 海正药业的重组人-鼠嵌合抗CD20单克隆抗体注射液治疗复发惰性CD20阳性B细胞非霍奇金淋巴瘤患者的I/Ⅱ期临床试验正在招募患者中。试验人数60人。
试验详情：重组人-鼠嵌合抗CD20单克隆抗体单次给药联合多次给药的 I期安全性、耐受性、药代动力学和药效学临床研究。招募病例数9-18例。试验用药：重组人-鼠嵌合抗CD20单克隆抗体注射液，规格10ml：100mg/瓶；静脉注射（IV），每周给药一次，低剂量组为250mg/m2，中剂量组为375mg/m2，高剂量组为500mg/m2。用药时程：连续给药4次，共计22天1个周期。主要终点指标：单次给药后35天，多次给药首次给药后35天，单次给药及多次给药剂量递增的安全耐受性，剂量限制性毒性（DLT）和最大耐受剂量（MTD)。2016年开展的单臂、非随机化开放的海正重组人-鼠嵌合抗CD20单克隆抗体注射液治疗复发惰性CD20阳性B细胞非霍奇金淋巴瘤患者的I/Ⅱ期临床试验，招募病例人数60人。试验用药：海正重组人-鼠嵌合抗CD20单克隆抗体注射液，静脉滴注，每周1次，每次250 mg/㎡（低剂量）、375 mg/㎡（中剂量）、500 mg/㎡（高剂量）、625 mg/㎡（最高剂量） mg/㎡；用药时程：连续4周。主要终点指标：诱导治疗后1个月（首次给药后第56±2天），肿瘤总缓解率(ORR)：比较完全缓解（CR）+部分缓解（PR）的受试者比例。 点评：该试验若按照biosimilar申报策略进行该试验，可不进行剂量摸索；为单臂试验未做与原研药的比对研究，并且为多次给药，同时样本量较小。疑为以新药申报模式所进行的I期、I/II期临床试验。神州细胞工程有限公司 2013年下半年完成SCT400连续多次给药治疗CD20阳性B细胞型NHL患者的多中心、开放I期安全性、耐受性、药代动力学和药效学临床研究，2015年开始启动多中心、随机、开放、阳性对照比较SCT400与利妥昔单抗在非霍奇金淋巴瘤患者的PK，PD和安全性的II期临床试验，试验人数80人，目前正在招募中。2016年启动随机、受试者设盲、多中心、阳性对照比较S-CHOP与R-CHOP在初治的CD20阳性的DLBCL患者中的有效性和安全性的III期临床试验，试验人数330人，目前正在招募中。试验详情：SCT400连续多次给药治疗CD20阳性B细胞型NHL患者的多中心、开放I期安全性、耐受性、药代动力学和药效学临床研究，招募病例数15人。试验用药：SCT400注射液，注射液；规格 50 mg(5 ml)/瓶；静脉滴注（IV),一周一次，每次250 mg/m2（低剂量组）、每次375 mg/m2（中剂量组）、每次500 mg/m2（高剂量组）；用药时程：连续用药共计4周。终点指标：连续4周给药期间及疗程结束后1周随访观察期，剂量限制性毒性（DLT）和最大耐受剂量（MTD)。多中心、随机、开放、阳性对照比较SCT400与利妥昔单抗在非霍奇金淋巴瘤患者的PK，PD和安全性的II期临床试验，招募病例数80例。试验用药：SCT400 IV, 375mg/㎡, 单次给药；对照药：美罗华，IV, 375mg/㎡, 单次给药。主要终点指标：自给药起3个月，SCT400和利妥昔单抗单次给药后的PK参数：AUC0~∞。随机、受试者设盲、多中心、阳性对照比较S-CHOP与R-CHOP在初治的CD20阳性的DLBCL患者中的有效性和安全性的III期临床试验，招募病例数330例。试验用药：S-CHOP， SCT400注射液，规格50mg(5ml)/瓶；静脉滴注（IV），在每周期的第1天给药，21天为一个周期；每次给药剂量375mg/m2； 用药时程：连续给药6个周期（或疾病进展）；对照用药： R-CHOP 美罗华，规格100mg（10ml）/瓶；静脉滴注（IV），在每周期的第1天给药，21天为一个周期；每次给药剂量375mg/m2； 用药时程：连续给药6个周期（或疾病进展）。主要终点指标：接受研究药物6个周期(18周)后，总缓解率。 点评：该临床试验设计较为合理。 3国内新适应症的拓展及新药申报策略的临床试验设计随着对利妥昔单抗不断深入的研发，越来越多的适应症被批准，FDA批准的适应症有： ·
Non-Hodgkin’s Lymphoma（NHL） 非霍奇金淋巴瘤·
Chronic Lymphocytic Leukemia（CLL） 慢性淋巴细胞白血病·
Rheumatoid Arthritis（RA）in combination with methotrexate in adult patients with moderately-to severely-active RA who have inadequate response to one or more TNF antagonist therapies 在中度至重度类风湿关节炎、对一个或多个TNF拮抗药治疗不充分的成年病人中联合甲氨蝶呤治疗类风湿性关节炎·
Granulomatosis with Polyangiitis(GPA)(Wegener’s Granulomatosis) and Microscopic Polyangiitis(MPA) in adult patients combination with glucocorticoids在成年患者中与糖皮质激素联合治疗多血管炎伴随的肉芽肿（韦氏肉芽肿病）和微观多血管炎（MPA） 由于美罗华在中国的适应症为冬亚型的非霍奇金淋巴瘤的治疗，因此，国内申报单位在获得类风湿性关节炎（RA）适应症的临床批件后，须以新药申报策略进行后续的临床试验设计。例如上海复宏汉霖的临床试验是这样设计的： 2015 年5月，公司HLX01获得了类风湿性关节炎（RA）的适应症临床批件，目前在Ⅰ期临床。试验详情：在类风湿关节炎受试者中评估HLX01和美罗华的药代动力学、药效动力学、安全性和疗效的随机、双盲、I/II期临床研究。招募病例数194人。试验组为HLX01注射液：单剂量1000 mg，在第1天和第15天进行静脉滴注；对照组为美罗华：单剂量1000 mg，在第1天和第15天进行静脉滴注。主要终点指标：从0时至无限时浓度-时间曲线下面积[AUC(0-inf)；μg.h/mL]，包括第一、二次用药。 点评：类风湿性关节炎适应症是美罗华在国内未被批准的适应症，因此按照新药策略进行申报，同时该适应症的设计需要较大的样本量。
C ONCLUSION
语利妥昔单抗，作为国内最看好的单抗品种之一，在罗氏2016H1主要产品销售额中，表现为29.52亿瑞士法郎。随着利妥昔单抗不断拓展的新适应症，销售额将继续攀升。据知名的医药咨询公司Igeahub推出的“2018年最畅销10大孤儿药”排行榜中，利妥昔单抗、来那度胺、依库珠单抗位列前三。目前国内生物制药公司布局该品种的爆发式热情，最终将使病患者受益于该品种biosimilar产品的陆续上市，并将出现群雄争鹿的竞争格局。
相信在众多科研工作者的努力下，肿瘤君终有一天会被消灭~但在此之前，小伙伴们还是要注意日常预防哦，健康饮食，强身健体，保持好心情，练就金钟罩铁布衫的同时，离女神/男神又近一步啦~
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作者：冯见
来源：生物谷
关键词：Apotex,生物类似物,Amgen,Filgrastim,生物药
日讯/生物股BIOON/-Apotex公司，一家正在全球激烈的生物仿制药市场中崛起的竞争者，日宣布， 受理公司产品Filgrastim非格司亭生物仿制药Grastofil™的申请，这是Amgen公司优保津的，由Apotex和Intas制药公司共同开发。
这已经是Apotex公司在美国通过由生物仿制药价格竞争和创新法案（BPCIA）创建的351(k)简化审批途径向FDA递交的第二个生物仿制药申请。Apotex公司之前通过351(k)简化申请途径提交的另一个产品是PEG化长效Filgrastim非格司亭制剂，目前也正处于FDA审评阶段。 Apotex公司也是至今唯一一家有两个在审（PEG-Filgrastim and Filgrastim)的幸运者。
Filgrastim非格司亭是Amgen公司应用重组DNA技术生产的甲硫氨酸人粒细胞集落刺激因子(Granulocyte colony-stimulating factor，G-CSF)，可促进造血辅助细胞增殖分化，明显增加外周血中性粒细胞数，并有增强粒细胞的功能，如对成熟的中性粒细胞可增强其吞噬活性和抗体依赖性细胞介导的细胞毒作用。根据Symphony Health Solutions公司的数据， Neupogen？( Filgrastim非格司亭) 2014年大约有10亿美金的销售额。
“我们非常高兴能够成为全球首先将高质量生物仿制药引入美国市场的公司之一”，Apotex 公司董事长和CEO Jeremy B. Desai 博士说，“对于40年来一直在全球范围内提供高质量、可负担性治疗药物的Apotex来说，进入美国医药的前沿领域是我们发展历史的分水岭事件”，他补充到，“对于患者，支付方和药物提供者来说，必将带来很明显的好处。我们致力于在促进美国公众获得可负担的救命药物以及大幅度降低美国医疗保障体系成本方面发挥关键的作用”。
该产品在美国将由Apotex的子公司ApoBiologix™全权进行销售。
今年年初，美国药物评审中心（CDER）举行的药物专家顾问委员会（ODAC）上，评议乐旗下山多士（Sandoz）公司申报的安进公司Neupogen的生物仿制药EP2006（欧洲商品名：Zarzio）的生物制剂许可申请（BLA）。经过听取山多士公司的申报报告、FDA评审员的评议报告和公众听证，专家小组以14：0的不记名投票结果一致推荐批准山多士的EP2006上市（美国商品名：Zarxio）。 可以预见，在不久的将来，美国市场上的Neupogen将有一番激烈的“搏斗“。
关于Apotex公司
Apotex Inc.成立于1974年，是加拿大最大的现代化制药企业，在全球拥有6000多名员工，从事药品的研制开发，生产制造与上市销售。APOTEX生产的奥贝品牌药品行销全球115个国家，享有国际声誉。
APOTEX拥有先进的生产设备，其生产能力已超过加拿大所有其它制药企业生产能力的总和。APOTEX生产200多个品种、2000多个规格和剂型，其中包括固体制剂、液体制剂、注射剂、搽剂及喷雾剂等，仅固体制剂的年生产能力即达180亿片/胶囊。
APOTEX每年投入年销售额的20%用于新药的研制和开发，其研发费用的投入列加拿大所有企业的前列，位居加拿大制药行业前列。新产品的研制和开发是APOTEX重要的发展方向，涉及血液科药物、心血管药物、抗生素、精神科药物和抗药物等多个领域。（）
Apotex Announces
Has Accepted For Filing its Biosimilar Application for Filgrastim (Grastofil™)
Company currently has two biosimilar applications (filgrastim [Neupogen™]) and pegfilgrastim [Neulasta™]) under active review by .
Apotex Inc.， a rising competitor in the global biosimilars market， announced today that， as of February 13th， 2015， the US Food and Drug Administration has accepted for filing the company's application for Filgrastim [Grastofil™]， a biosimilar version of Amgen's Neupogen™. This product has been jointly developed with Intas Pharmaceuticals Ltd.
This is the second follow-on biologic FDA submission for Apotex via the 351(k) abbreviated approval pathway created by the Biosimilar Price Competition and Innovation Act (BPCIA).& Apotex also has a 351k biosimilar application for the long acting pegylated formulation of filgrastim currently under FDA review.& Apotex is the only company to date to have two biosimilar filgrastim applications (pegfilgrastim and filgrastim) currently under active review by . Filgrastim is used to help cancer patients taking chemotherapies fight infections and fever by boosting white blood cell counts.& According to Symphony Health Solutions， Neupogen™ had approximately $1-billion in sales in calendar year 2014.
"We are very pleased to be at the forefront of companies who will introduce high quality biosimilar products into the US marketplace，" said Apotex President and Chief Executive Officer， Dr. Jeremy B. Desai. "Our entry into this new frontier of medicine in the United States is a watershed event in Apotex's 40 year history of providing quality， affordable medicines to patients in need around the globe，" Desai added. "The benefits for patients， payers and providers from biosimilars will be significant. We are dedicated to playing a leading role in the effort to increase the American public's access to more affordable versions of these life-saving therapies and generate substantial savings for the US health care system，" Desai added.
The product will be marketed in the United States by ApoBiologix™， a division of Apotex Corp.
About Apotex
Apotex is the largest Canadian owned pharmaceutical company with over 10，000 employees globally and with estimated sales of approximately $2 billion. The company's US headquarters is based in Weston， Florida. With its worldwide manufacturing sites， Apotex can produce up to 24 billion dosages per year. It produces 300 medicines in 4，000 dosages and formats that are exported to 115 countries. It has 500 products under development and will spend $2 billion over the next 10 years on research and development.
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中国的市场的确存在很大的隐患，干细胞行业是个专业性要求较高的行业
个体化医疗是未来医学研究与应用的趋势，而个体化治疗的关键在基于生物分子标志物的诊疗策略
中国疫苗市场的巨大潜力，吸引了世界排名最领先的跨国疫苗制造巨头前来淘金。Antibody Drug Conjugate model fast characterization by LC-MS following IdeS proteolytic digestion (PDF Download Available)
48 CitationsSee all >34 References
more authorsAbstractHere we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glyco-profiling and demonstration of the absence of additional conjugation was easily achieved.
As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.Discover the world's research14+ million members100+ million publications700k+ research projects
PAPER TYPEwww.landesbioscience.com mAbs
173mAbs 6:1, 173–184; January/February 2014; (C) 2014 Landes BioscienceREPORTIntroductionAntibody-drug conjugates (ADCs), or immunoconjugates aiming to combine the potency of cytotoxic drugs with the high specificity of a monoclonal antibody (mAb), are becoming increasingly important as new targeted therapies in oncology.1 Two ADCs, brentuximab vedotin (Adcetris(R)) and ado-trastu-zumab emtansine (Kadcyla(R)), have been recently approved by the US Food and Drug Administration, and more than 30 are cur-rently being investigated in clinical trials.2-5 To construct ADCs, highly potent cytotoxic agents are chemically attached to mAbs specific to tumor-related antigens with cleavable or non-cleavable linkers. Depending on the conjugation chemistries, different ADC structures have been developed. The drug payloads may be, for example, randomly attached to surface-exposed lysine residues distributed on both light and heavy chains (average of 80 to 95 per IgG), as illustrated by ado-trastuzumab emtansine.6 Alternatively, site-specific coupling to two or more of the eight cysteine residues involved in inter-chain disulfide bridges of chi-meric, humanized or human IgG1 after mild reduction may be used, as illustrated in the case of brentuximab vedotin.7 For the same purpose, the glycan moiety can be subjected to mild oxida-tion and used for site-selective conjugation via a hydrazone link-age.8 As further improvements for the third-generation ADCs, design of optimized antibodies (e.g., engineered to contain free surface-exposed cysteines for a site-specific drug linkage) and drug loading level optimization are investigated to maintain the natural favorable pharmacokinetics (PK) of chimeric, human-ized, and human IgGs. Site-specific conjugation to antibodies results in more homogeneous drug loading and avoids ADC sub-populations with altered antigen-binding caused by cross-linking in the CDRs or altered PK caused by cross-linking at Fc domain binding sites of FcRn. Several such attempts have recently been described, including the addition of two cysteines in the antibody variable domains9 or in the constant domains.10-12Conjugation of drugs to mAbs increases the structural com-plexity of the resulting molecule, which triggers the need for improved characterization methods.13 Antibody-fluorophore conjugates (AFCs) using the same linkers and conjugation chem-istries as ADCs, but with a non-toxic cargo, are valuable molecu-lar tools for mechanistic studies and PK evaluation as recently illustrated in several reports (e.g., Alexa48814 and Alexa350,15 *Correspondence to: Elsa Wganer; Email: elsa.wagner@pierre-fabre.com; Alain Be Email: alain.beck@pierre-fab</Submitted: 09/21/13; Revised: 10/09/13; Accepted: 10/10/13http://dx.doi.org/10.4161/mabs.26773Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestionElsa Wagner-Rousset1,*, Marie-Claire Janin-Bussat1, Olivier Colas1, Melissa Excoffier1, Daniel Ayoub1, Jean-Fran?ois Haeuw1,
Ian Rilatt2,
Michel Perez2, Nathalie Corva?a1, and Alain Beck1,*1IRPF; Centre d’Immunologie Pierre F St Julien-en-Genevois, France; 2IRPF; Centre de Recherche Pierre Fabre; Castres, FranceKeywords: ADC, antibody-drug conjugates, AFC, antibody-fluorophore conjugates, DAR, multimers, IdeS, N-linked glycosylation, mass spectrometryHere we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first character-ized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glyco-profiling and demonstration of the absence of additional conjugation was easily achieved.As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analyti-cal method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.
Volume 6 Issue 1or biotin16). Here, we report the design and production of an AFC as a non-toxic ADC model to extend the analytical plat-form for optimization of next-generation ADCs (OptimADCs). Our AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab. Trastuzumab is frequently used as a reference, both as a naked antibody and as an ADC.17,18 DSEA-linker maleimide payload (Fig. 1B) was designed and synthesized (Fig. S1) to mimic the chemistry and the linker of brentuximab vedotin and the majority of ADCs in clinical trials (Fig. 1A).As proof of principle, the trastuzumab-mc-DSEA conjugate was initially analyzed by the current analytical methods used for antibody conjugated to maleimidocaproyl valine-citruline mono-methylauristatin E (vc-MMAE) (Fig. 1A)19 to demonstrate the comparability of the resulting profiles,20 including size-exclusion chromatography (SEC) (Fig. 2), sodium dodecyl sulfate-polyacryl-amide Gel (SDS-PAGE) (Fig. 3), capillary electrophoresis sodium dodecyl sulfate (CE-SDS) (Fig. 4), hydrophobic interaction chro-matography (HIC) (Fig. 5), and high performance liquid chroma-tography with UV and mass spectrometry detection (HPLC-UV/MS) in both denaturating conditions and native (Fig. 6).21-23Subsequently, digestion of the AFC by immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), followed by LC-MS was investigated. IdeS specifically cleaves immunoglobu-lin G under its hinge domain.24-26 In his 2012 published notes, Chevreux et al. described the advantages of IdeS proteolytic digestion prior to LC-MS of recombinant mAbs.27 Under reduc-ing conditions, IdeS digestion of mAbs results in three poly-peptide chains of around 25 kDa each. With minimal sample preparation, and within a single shot analysis, the method pro-vides efficient LC and MS resolution that potentially results in relevant information on N-glycan profiling, charge state variants such as C-terminal lysine truncation, pyroglutamylation, oxida-tion and product cleavages.The use of IdeS is becoming increasingly popular for the fast characterization of antibody by mass spectrometry,27, 2 8 including correct sequence assessment,29,30 antibody Fab and Fc glyco-pro-filing,31 biosimilar comparability studies and Fc-fusion protein studies.32 Here, we report and discuss the potential of IdeS for ADC and AFC fast characterization using the trastuzumab-mc-DSEA conjugate and allowing glycoprofilling which may be important to retains the effector function of the naked parent antibody.33 ResultsThe raw trastuzumab-mc_DSEA analyzed by SEC (Fig. 2A) showed the presence of two main peaks interpreted as multimeric (69.5%) and monomeric species (30.5%). Both populations were separated by a preparative SEC and further characterized by Figure?1. Structures of (A) linker-cytotoxic (mc_MMAE) and (B) linker-fluorescent (mc_DSEA) payloads.
www.landesbioscience.com mAbs
175SDS-PAGE (Fig. 3), CE-SDS (Fig. 4), HIC (Fig. 5), native mass spectrometry (Fig. 6), and liquid chromatography coupled to electrospray time of flight mass spectrometry MS (LC-ESI-TOF) after IdeS digestion and reduction. We investigated the structural characterization of both purified populations to establish a poten-tial relationship between aggregation of the AFC and its average dye-to-antibody ratio (DAR). The SEC chromatogram of the purified monomeric products performed at an analytical scale is presented in Figure 2B and shows a main peak of monomers at
30.3 min (96.3%) and dimers (3.7%).Monomeric and multimeric fractions of the AFC vs. non-conjugated trastuzumab were analyzed by SDS-PAGE under non-reducing and reducing conditions (Fig. 3). Unreduced trastuzumab displays a main band at ~150 kDa that corresponds to the H2L2 form and another lighter band of lower molecular weight likely to correspond to the molecule lacking one light chain, which is commonly described as a result of the produc-tion process. The pattern of the non-reduced monomeric AFC fraction is distributed as following: six bands of apparent molecu-lar weight around 150, 125, 100, 75, 50, and 25 kDa, which fit to H2L2 and H2L, H2, HL, H and L, respectively, each carry-ing payloads. Indeed, because some of the interchain disulfide bridges are disrupted by the conjugation, the structure of the typical heterodimeric mAb (H2L2) is no longer maintained in the presence of SDS.The profile of the non-reduced multimeric AFC fraction looks different in its distribution because only H2, HL, H, L plus payloads are present. The intensity of each band is also differ-ently distributed with less intense H2 and HL bands with respect to H and L. Altogether, these results highlight a different pay-load distribution between monomeric and multimeric fractions, with a higher level of conjugation for the multimeric AFC. The SDS-PAGE pattern under reducing conditions confirms these observations. Indeed, the apparent molecular weight of the light and heavy chains increases between unconjugated trastuzumab, monomeric AFC and multimeric AFC, respectively. The heavy chain, which has three cysteines capable of forming interchain disulfides, may carry up to three payloads, resulting in an increas-ing distribution in our case. On the other hand, the light chain may carry a maximum of one payload because it only has one interchain disulfide-forming cysteine. This does not fit with the slightly elevated molecular weight observed for the light chain of the multimeric fraction compared with the monomeric one, and may be the result of over-payload conjugation. This will be discussed in the following sections.IdeS digestion of an ADC/AFC followed by a reduction step can generate seven fragments as illustrated in Figure 7. Fd and LC exist as naked or conjugated forms carrying up to 3 payloads (depending on the number of conjugated interchain disulfide-forming cysteines) plus Fc/2, which is theoretically free of pay-load and carrying the glycosylation heterogeneity. Figure 8 shows the UV chromatogram at 280 nm resulting from the elution of trastuzumab that was digested with IdeS then reduced. The three resulting fragments were well-resolved and identified accord-ing to their MS profile (Fig. 8B-D; Table 1). The first eluting peak at 11.3 min corresponds to the Fc/2 fragments reflecting the pattern of trastuzumab being N-glycosylated by biantennary glycans. Mainly agalactosylated fucosylated (G0F) and monoga-lactosylated fucosylated (G1F) species were detected (at 25 237 and 25 399 Da respectively), whereas only a small amount of afucosylated (G0) and bigalactosylated fucosylated (G2F) forms were found (low intensity signals at 25 091 and 25 560 Da). This is as expected for an antibody produced in a CHO cell line. Eluting at 13.6 min and 17.5 min respectively, we identified LC Figure?2. Purification of trastuzumab-mc_DSEA. (A) Preparative SEC. (B) SEC analysis of monomeric AFC.
Volume 6 Issue 1and Fd fragments with low-intensity Na adducts and loss of water molecule for the Fd fragment.Figure 9A depicts the UV chromatogram at 280 nm resulting from the elution of trastuzumab-mc_DSEA digested with IdeS then reduced. Seven species were resolved and identified by the mass measurement (Fig. 9B-H; Table 2). As previously described for unconjugated trastuzumab, the Fc/2 fragments elute at
11.5 min with their typical glycoform distribution. Eluting at 14.3 and 16.6 min, within two completely resolved chromato-graphic peaks, we observed L0 and L1 differing from each other by a mass increment of 1,005 Da. This corresponds to the conju-gation of one mc-DSEA payload to the cysteine residue normally responsible for the formation of an inter-chain disulfide with the trastuzumab heavy chain. In a similar manner, we detected the Fd fragment from IdeS digestion coupled with 0, 1, 2, or 3 payloads and eluting at 18.1, 20.5, 25.9, and 30 min. The mass spectra look quite homogeneous, with low-intensity Na adducts (+21 Da) and some loss of a water molecule. The number of addi-tional payloads detected on the Fd fragment fits with the number of possible free thiol residues oxidized during preparation of the AFC (one cysteine linking the LC and two cysteines in the hinge area). In our chromatographic conditions, Fd1 and Fd2 are each split in two UV peaks sharing the same mass, which can be inter-preted as positional isomers.Figure?3. SDS-PAGE of the purified monomeric and multimeric forms of trastuzumab-mc_DSEA in non-reducing (NR) and reducing (R) conditions.Figure?4. CE-SDS-PAGE of the purified (A) monomeric and (B) multimeric forms of trastuzumab-mc_DSEA in non-reducing (NR) and reducing (R) conditions.
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177A similar LC-MS experiment was performed on the IdeS-digested, reduced multimeric trastuzumab-mc_DSEA. Chromatogram and mass spectra are presented in Figure 10 (see also Table 3). The resulting profile looks qualitatively and quan-titatively different than the previously studied monomeric frac-tion. Indeed, co-eluting together with the Fd1 fragment between 19.7 and 21.1 min, we detect some light chain carrying two pay-loads (L2) with a mass of 25 452 Da. An additional species at 34.5 Figure?5. Drug loading distribution and average DAR calculation by HIC. (A) Monomeric trastuzumab-mc_DSEA. (B) Multimeric trastuzumab-mc_DSEA. Average DAR is calculated by quantifying the various loaded forms based on the peak areas (A) of the UV chromatogram at 210 nm.Figure? 6. Drug loading distribution and average DAR calculation by native mass spectrometry.Figure?7. Generation of seven fragments of near 25 kDa after IdeS digestion and DTT reduction of trastuzumab-mc_DSEA.
Volume 6 Issue 1min corresponds to Fd4 (29 402 Da). As predicted by the SDS-PAGE results, the multimeric fraction seems to contain highly conjugated AFC with an unexpected payload distribution.To confirm this assumption, we further investigated the pay-load distribution, the results of which are presented in Figure 11. By analysis of the peak areas after integration, we determined the percentage of L0 and the conjugated forms for the light chain, fragments, as well as conjugation of the Fd region (Fd0, Fd1, Fd2, Fd3 and Fd4). The highest loaded fragments were found in higher concentration in the multimeric fraction compared with the monomeric fraction. Correspondingly, the opposite was true for the less conjugated forms. Thus, whereas L0 and L1 repre-sent 32% and 68% of the light chain in the monomeric fraction, respectively, they are 12% and 75% in the multimeric fraction, with 13% of L2 present. Similarly, Fd forms are represented as following in the monomeric fraction: Fd0 (16%), Fd1 (53%), Fd2 (26%), and Fd3 (5%), whereas we calculated Fd1 (3%), Fd2 (16%), Fd3 (72%) and Fd4 (9%) for the multimeric one.Average DAR was estimated from the latter calculated distri-butions as described in Figure 9. An average value of 3.8 payloads per antibody was determined for monomeric AFC whereas the average for multimeric AFC was 7.8.DiscussionBroadening the application to mAbs first described by Chevreux et al.,27 the use of IdeS for characterization of antibody conjugates by LC-MS has been described. The method has all the advantages as for unconjugated mAbs, with the added bonus of access to supplementary structural information specific to AFCs or ADCs. Within a simple and fast sample preparation, the method generates fragments of reasonable size (25 kDa). They are easily ionized and analyzed by ESI-TOF-MS, and in a better manner than for the heavy chain resulting from a single reduction process, for example. In addition, the approach can also monitor variants such as C-terminal lysine truncation, pyroglutamylation, oxidation and degradation products. Whereas LC and Fd frag-ments bear information related to conjugation and thus to ADC loading, Fc/2 affords accurate profiling of N-glycosylation, which is important because an ADC keeps the same effector functions as the naked antibody, as reported for trastuzumab and ado-trastuzumab emtansine.34 On the other hand, the LC-MS profile of the Fc/2 fragment should be unchanged throughout Figure?8. LC-UV-MS of trastuzumab after IdeS digestion and reduction. (A) UV chromatogram at 280 nm. (B?D) mass spectra of peaks eluting at 11.3, 13.6 and 17.5 min, respectively.Table?1. Mass assignment of species eluting in the chromatogram pre-sented in Figure?8.Time (min) AssignmentTheoretical masses (Da)Experimental masses (Da)11.3 Fc/2-G0 25 086 25 091Fc/2-G0F(-H2O) 25 214 25 218Fc/2-G0F 25 232 25 237Fc/2-G0F(+Na) 25 254 25 255Fc/2-G1F 25 394 25 399Fc/2-G2F 25 556 25 56013.6 LC 23 439 23 443LC(+Na) 23 461 23 46217.5 Fd (-H2O) 25 366 25 364Fd 25 384 25 383Fd(+Na) 25 406 25 401
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179the ADC production process and could therefore be used as an internal reference, since the various loaded forms of Fd and LC fragments can be quantified relative to the proportion of Fc/2.As depicted in the chromatographic profiles of our IdeS-reduced AFC, Fd1 and Fd2 split into two peaks. Indeed, the process of conjugation yields a controlled but heterogeneous population in terms of the number of payloads linked and the position of the linkage on the interchain disulfide cysteine residues. Under the reducing conditions of the conjugation reaction, only the Fd fragment carrying three of those cysteines may exhibit such positional isomers. In a remarkable manner, Le et al. developed a mathematical approach to quantify the proportion of each, and validated their systems by crossing calculated values with experimental results for several antibodies conjugated to vcMMAE.20 They showed that whatever the average DAR (ranking from 2.0 to 5.5), the most represented ADC isomers were so-called DAR2f, DAR4ff and DAR6fhh, where f and h are conjugation sites (f refers to the disulfide bridge between light and heavy chain and h to one in the hinge region). From these observations, we may assume that the most represented isomers in our chromatogram correspond to Fd1f and Fd2fh, whereas smaller peaks would be the Fd1h and Fd2hh isomers. These Figure?9. LC-UV-MS of monomeric trastuzumab-mc_DSEA after IdeS digestion and reduction. (A) UV chromatogram at 280 nm. (B?H) Mass spectra of peaks eluting at 11.5, 14.3, 16.6, 18.1, 20.5, 25.9 and 30 min, respectively.
Volume 6 Issue 1observations are also in agreement with data reported by H. Liu and colleagues on the ranking of the susceptibility of disulfide bonds in human IgG1 antibodies.34Finally, the method can be applied routinely to the determi-nation of payload distribution and calculation of average DAR, which has not been described until now. In our case, it allowed comparison between two SEC fractions purified from the same production batch targeting an average DAR of 4. We highlighted a very significant difference between both samples because the multimeric fraction consists of highly loaded forms (average DAR of 7.8), whereas the monomeric fraction more closely resembles the targeted distribution (average DAR of 3.8). In our case, there was a correlation between the number of conjugated payloads and the tendency for aggregation. We have also observed this for other mAbs and other payloads (data not shown). The hydrophobicity of the payload might also affect the ratio of resulting aggregates. By LC-MS analysis, we described fragments with more payloads than available interchain disulfide-forming cysteines (Fd4, L2), showing that the conjugation reaction may lead to uncontrolled alkylation. We presume that intrachain disulfide bridges were also reduced and conjugated during the reaction process, show-ing that the mild reduction conditions are somehow difficult to control. An extensive analysis by LC-MS peptide mapping is in progress to further investigate the position of payloads.In summary, as for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference ana-lytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer of ADCs, and to define critical quality attributes in a quality-by-design approach.Material and MethodsAntibody and linker-payload production and purificationThe trastuzumab used in this study is the European Medicines Agency-approved version and formulation
(21 mg/mL). The linker-fluorophore payload was designed to mimic the linker-drug most frequently used in ADC clinical trials. The synthesis is briefly reported in the supplemental material. It consists of maleimide-caproic acid dansyl sulfonamide ethyl amine (mc_DSEA, see structure Fig. 1) with a valine-citruline linker that mimics the cytotoxic agent and linker conjugated to mAbs through reduced interchain cysteine via the maleimide function.Mild reduction of trastuzumab and coupling of DSEA-linker were performed as previously described.19 Briefly, trastuzumab was reduced with 2.75 equivalents of TCEP in 10 mM borate pH 8.4 buffer containing 150 mM NaCl and 2 mM EDTA for
°C. The concentration of free thiols was determined by using the Ellman’s reagent with l-cysteine as standard, typically resulting in around 5 thiols per antibody. To target a DAR of 4, the partially reduced trastuzumab was then alkylated with 2 equivalents of DSEA-linker per thiol in the same buffer for 1 h at room temperature. N-acetyl-cysteine (1.5 equivalents / DSEA-linker) was used to quench any unreacted DSEA-linker. The AFC was purified by size exclusion chromatography on a Superdex
200 pg column (GE Life Sciences) eluted with 25 mM histidine pH 6.5 buffer containing 150 mM NaCl, by using an AKTA Avant biochromatography system (GE Life Sciences). Fractions corresponding to AFC monomers and multimers were collected and concentrated. The protein concentration of the monomeric and multimeric AFC solutions was determined by BCA with bovine immunoglobulins as standard. The labeled antibodies Table?2. Mass assignment of species eluting in the chromatogram pre-sented in Figure?9.Time (min) AssignmentTheoretical masses (Da)Experimental masses (Da)11.5 Fc/2-G0 25 086 25 089Fc/2-G0F(-H2O)25 214 25 216Fc/2-G0F 25 232 25 235Fc/2-G0F(+Na)25 254 25 255Fc/2-G1F 25 394 25 397Fc/2-G1F(+Na)25 416 25 418Fc/2-G2F 25 55625 55914.3 L0 23 439 23 442L0(+Na) 23 46123 46316.6 L1 24 444 24 447L1(+Na) 24 46624 46818.1 Fd0 (-H2O) 25 366 25 362Fd0 25 384 25 383Fd0(+Na) 25 40625 40420.5 Fd1(-H2O) 26 371 26 368Fd1 26 389 26 388Fd1(+Na) 26 41126 40825.9 Fd2(-H2O) 27 376 27 373Fd2 27 394 27 392Fd2(+Na) 27 41627 41330.0 Fd3(-H2O) 28 379 28 378Fd3 28 397 28 397Fd3(+Na) 28 419 28 418Fd3(+2Na) 28 441 28 442
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181were further analyzed by SDS-PAGE on a 4–15% gradient gel (BioRad) under reducing and non-reducing conditions. The gel was stained with Coomassie blue. For analytical SEC, an Agilent HPLC system (Les Ulis) was used. Samples were analyzed on a Superdex 200 GL column (10 × 300 mm, GE Life Sciences) at room temperature. They were eluted at a flow rate of 0.4 ml/min with 25 mM histidine pH 6.5 buffer containing 150 mM NaCl as mobile phase. The elution was monitored at 280 nm.CE-SDS characterizationThe buffer of 100 ug of ADC sample was exchanged with SDS-MW sample buffer by using the Microcon YM30 centrifuge filter unit to have a final volume at 95 uL. Two milliliters of internal standard 10kDa were added with 5 uL of 250 mM iodo-acetamide for non-reduced sample or 5 uL of 2-mercaptoethanol for reduced sample. All tubes were heated at 70
°C for 10 min and centrifuged at 12000 g for 6 min. Samples were then ana-lyzed on ProteomeLab PA800 (Beckman) equipped with a UV detector. The detection was conducted at 220 nmHIC characterizationHIC was performed using a liquid chromatography system (Alliance HPLC) coupled to a detector UV (2489 dual absor-bance). Analyses were realized on a TSKButyl-NPR, 2.1 ×
Figure?10. LC-UV-MS of multimeric trastuzumab-mc_DSEA after IdeS digestion and reduction. (A) UV chromatogram at 280 nm. (B?H) Nass spectra of peaks eluting at 11.5, 14.1, 16.3, 19.7–21.1, 24.5–25.4, 29.3 and 34.5 min, respectively.
Volume 6 Issue 1Figure? 11. Drug loading distribution and average DAR calculation. (A) Unconjugated trastuzumab. (B) Monomeric. (C) Multimeric trastu-zumab-mc_DSEA, respectively. Average DAR is calculated by quanti-fying the various loaded forms based on the peak areas (A) of the UV chromatogram at 280 nm.4.6 mm column (Tosoh bioscience) set at room temperature. The mobile phase A consisted of 1.5 M ammonium sulfate, 25 mM potassium phosphate pH7.0, and the mobile phase B consisted of a mixture 25 mM potassium phosphate and 25% isopropanol at pH7. Separation was obtained with a linear gradient of 10–100% B over 12 min at flow rate of 0.8 mL/min. Analysis were obtained by injecting 20 ug of sample diluted at 1 mL/min with mobile phase A and integrating the UV area at 210 nm for each species.Native mass spectrometry characterizationPrior to native mass spectrometry experiments, an aliquot of 300 ug monomeric trastuzumab-mc_DSEA was deglycosylated by adding 0.6 uL PNGaseF (New England Biolabs) followed by incubation overnight at 37
°C. Thirty ug of deglycosylated sample were injected on a PolyHYDROXYETHYL A(TM) column (PolyLC), 150 × 1 mm, 5 um, 300 A°. Elution from the column was performed within a 6 min isocratic step at 0.1 mL/min with 200 mM ammonium acetate solution buffered at pH7. During sample elution, chromatography was coupled online with an electrospray time-of-flight mass spectrometer (LCT PremierTM, Waters) operating in V positive ion mode with a capillary volt-age of 3000 V and a sample cone voltage of 40 V. Acquisitions were performed on the mass range m/z 1000–12000 with 1 s scan time. Native desalting conditions maintain the intact biva-lent structure of the AFC. Well-resolved peaks were sufficient for confirmation of the identity of the AFC (0, 2, 4, 6, 8 payloads) and to determine the relative distribution of the drug-loaded spe-cies. The average DAR deduced from those measurements is 4.4.IdeS digestionTwenty UI of IdeS enzyme were added to 20 ug of AFC solu-tion following the instructions of the enzyme kit (FabRICATOR Kit, Genovis). The mixture was then incubated at 37
°C for 30 min. Before reduction, the digested sample is diluted twice with a buffer containing 6 M guanidine-HCl, 2 mM EDTA, TRIS-HCl 0.1 M. DTT is added to reach a final concentration of
10 mM. After 45 min incubation at 56
°C the reaction is quenched with 1 uL acetic acid.LC-MS analysisReverse-phase high performance liquid chromatography (RP-HPLC) was performed using an ultrahigh-performance liquid chromatography system (Acquity UPLC, Waters) cou-pled to an electrospray time-of-flight mass spectrometer (LCT PremierTM, Waters). A volume equivalent to 8 ug of sample prepa-ration was injected on a PLRP-S 1000A°, 8um, 150 × 2.1 mm column (Agilent) set at 80
°C. The gradient was generated at a Table?3. Mass assignment of species eluting in the chromatogram pre-sented in Figure?10.Time (min) AssignmentTheoretical masses (Da)Experimental masses (Da)11.5 Fc/2-G0 25 086 25 089Fc/2-G0F(-H2O) 25 214 25 214Fc/2-G0F 25 232 25 236Fc/2-G0F(+Na) 25 254 25 258Fc/2-G1F 25 394 25 398Fc/2-G1F(+Na) 25 416 25 420Fc/2-G2F 25 556 25 56114.1 L0 23 439 23 443L0(+Na) 23 461 23 46516.3 L1 24 444 24 448L1(+Na) 24 466 24 46919.7–21.1 L2 25 453 25 452L2(+Na) 25 474 25 472Fd1 26 389 26 38724.4–25.4 Fd2(-H2O) 27 376 27 371Fd2 27 394 27 393Fd2(+Na) 27 416 27 41629.3Fd3(-H2O)Fd3Fd3(+Na)28 38028 39728 41928 37828 39828 41830.0 Fd4 29 404 29 402Fd4(+Na) 29 426 29 422
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183flow rate of 0.25 mL/min using 0.05% trifluoroacetic acid (TFA) for mobile phase A and acetonitrile containing 0.05% TFA for mobile phase B. B was raised from 5% to 30% in 8 min and to 50% in an additional 40 min followed by a 2 min washing step at 95% B and a 10 min reequilibration period. Elution was monitored spectrophotometrically at 280 nm. The LCT Premier was operated in W positive ion mode with a capillary voltage of 3000V and a sample cone voltage of 120 V. Acquisitions were performed on the mass range m/z 1000–4000 with a 1 s scan time. Calibration was performed using the singly charged ions produced by a 2 mg/L solution of cesium iodide in 2-propanol/water (1/1). Data analysis was performed with MassLynx 4.0 (Waters).Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.AcknowledgmentsWe would like to thank Laura Morel-Chevillet, Anne Viaud, and Anne Humbert for their technical support, Thierry Champion for structure modeling, Amandine Boeuf and Christine Klinguer-Hamour for helpful discussions, and Claire Catry for her assistance.Supplemental MaterialsSupplemental materials may be found here: www.landesbioscience.com/journals/mabs/article/26773References1.
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CitationsCitations48ReferencesReferences34ArticleJun 2017J Chrom BArticleJun 2017ArticleFeb 2017J ChromShow moreArticleApril 2016 · Analytical Chemistry · Impact Factor: 5.64A recombinant monoclonal antibody with trisulfide bonds and cysteinylation was thoroughly characterized in the current study. Trisulfide bonds and cysteinylation were first detected when the recombinant monoclonal antibody was analyzed by LC-MS to determine the molecular weights of the intact antibody and its F(ab&#x27;)2 fragment generated from IdeS digestion. LC-MS analysis of non-reduced tryptic... ArticleJanuary 2012 · Journal of biomolecular techniques: JBTArticleApril 1994 · The FASEB Journal · Impact Factor: 5.04ArticleData provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. Publisher conditions are provided by RoMEO. Differing provisions from the publisher&#x27;s actual policy or licence agreement may be applicable.This publication is from a journal that may support self archiving.