CN116712390A - 一种高浓度高稳定性的抗体制剂及其制备方法 - Google Patents
一种高浓度高稳定性的抗体制剂及其制备方法 Download PDFInfo
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- CN116712390A CN116712390A CN202310976752.0A CN202310976752A CN116712390A CN 116712390 A CN116712390 A CN 116712390A CN 202310976752 A CN202310976752 A CN 202310976752A CN 116712390 A CN116712390 A CN 116712390A
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Abstract
本发明提供一种高浓度高稳定性的抗体制剂及其制备方法,属于抗体制剂领域,所述的抗体制剂中包括一种抗C5抗体,轻链序列为SEQ ID NO.1,重链序列为SEQ ID NO.2或其长效改造序列;还包括缓冲体系:20‑50mM组氨酸‑盐酸组氨酸、70‑110mM精氨酸、25‑50mM氯化钠和0.03%PS80。常规抗体制剂浓度一般在100mg/mL以下,本发明缓冲液体系抗体浓度达到200mg/mL左右。且本发明的抗体制剂表现出了良好的长期稳定性、亲和力和生物利用度。
Description
技术领域
本发明属于抗体制剂领域,具体涉及一种高浓度高稳定性的抗体制剂及其制备方法。
背景技术
随着生物抗体药治疗的发展,对于静脉内或者皮下高浓度给药需要对有效成分进行高度浓缩(约20mg/mL至约100-150mg/mL,甚至200mg/mL或更高)。在对单次给药量较大的慢性疾病,如PNH(阵发性睡眠性血红蛋白尿症,表现为慢性血管内溶血)的治疗领域,为了实现病人的自主用药,提升病人用药的便捷性和治疗依从性,需要开发给药体积小而浓度高的抗体制剂。然而,抗体药物在高浓度浓缩过程中,会产生如粘度增大、蛋白聚集及电荷异质性改变的问题,会引起活性抗体活性下降、免疫原性增强等风险。因此,需要改进抗体药物的制剂以增加抗体的溶解度,降低粘度,并解决蛋白聚集、电荷异质性改变的问题。
对于抗体药物制剂的改进主要包括以下几个方面:
I.制剂中的抗体;
II.钙和乙酸盐或者缓冲液:
通过向所述制剂中加入钙盐和/或乙酸盐(和/或缓冲液)降低抗体制剂的粘度:测定方法在加入钙盐和/或乙酸盐(和/或缓冲液)之前和之后测定所述蛋白制剂的粘度;
III.制剂中的赋形剂:
赋型剂能够赋予或增强蛋白制剂的稳定性,以及一定程度上影响药物的功效和免疫源性,因此需要开发具有稳定性、安全性的赋型剂的蛋白制剂。赋形剂可以是缓冲剂、稳定剂或表面活性剂等。
EA5单抗是一种特异性靶向补体C5的人源化单克隆抗体。补体C5在酶切后可转化为C5a和C5b,其中C5a为重要的促炎分子,C5b可继续形成C5b-9,即导致细胞打孔或裂解的攻膜复合物(MAC)。攻膜复合物在 PNH患者红细胞表面沉积可引起红细胞裂解,即PNH的主要临床表现。EA5单抗可阻断C5裂解,防止C5a和C5b-9的形成,因此可用于有效治疗PNH。另外对于aHUS、gMG等由补体末端激活引起的疾病同样有较好的治疗效果。
PNH(阵发性睡眠性血红蛋白尿症)是一种罕见且危及生命的造血干细胞克隆性疾病,患病率约为百万分之十三,临床主要表现为溶血性贫血、血红蛋白尿症、静脉血栓形成、骨髓造血功能衰竭以及平滑肌功能障碍等。补体***攻击红细胞造成的血管内溶血是PNH发病和死亡的核心因素。CD59为红细胞表面最重要的补体攻膜复合物(MAC)抑制因子,PNH患者由于位于X染色体(Xp22.1)的磷脂酰肌醇聚糖(PIG-A)基因发生获得性体细胞突变,导致CD59因糖基磷脂酰肌醇(GPI)合成缺陷不能结合到红细胞的细胞膜上,最终导致抑制补体通路活化的功能丧失。PNH的主要治疗手段包括补体抑制剂、糖皮质激素和异基因造血干细胞移植等,其中补体C5抑制剂Soliris(Eculizumab)或Ultomiris(Ravulizumab)为PNH的标准治疗,可有效控制血管内溶血,较少血栓形成,并提高长期生存率,但上述药品价格昂贵且需要静脉注射,患者需定期入院输注,对生活带来较大不便。因此,开发安全有效的皮下注射补体C5抑制剂用于PNH病人的治疗意义重大。
PNH为典型的补体激活引起的疾病,EA5抗体对其他由补体激活引起的疾病同样具有较大的治疗潜力,如类风湿性关节炎、抗磷脂抗体综合征、狼疮性肾炎、缺血再灌注损伤、非典型溶血性***综合征(aHUS)、典型溶血性***综合征、阵发性夜间血红蛋白尿(PNH)、致密物沉积病、视神经脊髓炎、多灶性运动神经病、多发性硬化、黄斑变性、HELLP综合征、自发性流产、血栓性血小板减少性紫癜、寡免疫性血管炎、大疱性表皮松解症、复发性流产、创伤性脑损伤、心肌炎、脑血管病症、周围血管病症、肾血管病症、肠系膜/肠血管病症、血管炎、过敏性紫癜性肾炎、全身性红斑狼疮相关血管炎、类风湿性关节炎相关血管炎、免疫复合血管炎、高安氏病、扩张型心肌病、糖尿病血管病变、川崎病、静脉空气栓塞,支架置入、冠状动脉旋磨术、经皮腔内冠状动脉成形术后再狭窄,重症肌无力、冷凝集素病、皮肌炎、阵发性寒冷性血红蛋白尿、抗磷脂综合征、格雷夫斯病、动脉粥样硬化、阿尔茨海默氏病、全身性炎性反应败血症、败血性休克、脊髓损伤、肾小球肾炎、移植排斥、桥本甲状腺炎、I型糖尿病、银屑病、天疱疮、自身免疫性溶血性贫血、特发性血小板减少性紫癜、古德帕斯彻综合征、迪戈斯病和灾难性抗磷脂综合征。
发明内容
为了解决上述问题,本发明提供了一种抗C5抗体和其制剂,优化了抗体的分子设计以及制剂的缓冲体系,目的在于提高该抗体制剂的整体稳定性。
本发明中,“PS80”指聚山梨酯-80,又称吐温-80。
本发明中,“组氨酸-盐酸组氨酸”指组氨酸钠+盐酸组氨酸缓冲液。
一方面,本发明提供了一种抗C5抗体。
所述的抗C5抗体为改造抗体,轻链序列为SEQ ID NO.1,重链序列为SEQ ID NO.2或其长效改造序列。该抗体分子通过工程化Fc区域,提高抗体的溶解度和稳定性;提高抗体等电点,以避免抗体在下游工艺中的损失;同时提高抗体与FcRn的亲和力,以实现更长的体内半衰期。
优选地,所述的重链序列的长效改造序列为SEQ ID NO.3。
另一方面,本发明提供了包含前述抗C5抗体的抗体制剂。
所述的抗C5抗体含量为0-200mg/L,优选为160-200mg/mL,进一步优选为180mg/mL。
所述的抗体制剂中还包括缓冲体系。
所述的缓冲体系中包括:20-50mM组氨酸-盐酸组氨酸、70-110mM精氨酸、25-50mM氯化钠和0.03% PS80。
所述的组氨酸-盐酸组氨酸的含量可以选自:20-30mM、30-40mM、40-50mM、25-35mM、25-45mM。
所述的精氨酸的含量可以选自:70-80mM、80-90mM、90-100mM、100-110mM、70-90mM、80-100mM、90-110mM、100-110mM。
所述的氯化钠的含量可以选自:25-30mM、25-35mM、30-50mM、40-50mM、35-40mM、25-45mM、30-45mM。
优选地,所述的缓冲体系中包括:20mM组氨酸-盐酸组氨酸、70-110mM精氨酸、25-50mM氯化钠和0.03% PS80。
更优选地,所述的缓冲体系中包括:20mM组氨酸-盐酸组氨酸、80mM精氨酸、25-50mM氯化钠和0.03% PS80。
进一步优选地,所述的缓冲体系中包括:20mM组氨酸-盐酸组氨酸、80mM精氨酸、50mM氯化钠和0.03% PS80。
所述的缓冲体系的pH为6.0-6.5。
优选地,所述的缓冲体系的pH为6.0-6.3。
进一步优选地,所述的缓冲体系pH为6.3。
本发明同时提供抗体制剂中所用缓冲体系。
又一方面,本发明提供了前述的抗C5抗体或抗体制剂在制备药物中的应用。
所述的药物适应症选自:类风湿性关节炎、抗磷脂抗体综合征、狼疮性肾炎、缺血再灌注损伤、非典型溶血性***综合征(aHUS)、典型溶血性***综合征、PNH、致密物沉积病、视神经脊髓炎、多灶性运动神经病、多发性硬化、黄斑变性、HELLP综合征、自发性流产、血栓性血小板减少性紫癜、寡免疫性血管炎、大疱性表皮松解症、复发性流产、创伤性脑损伤、心肌炎、脑血管病症、周围血管病症、肾血管病症、肠系膜/肠血管病症、血管炎、过敏性紫癜性肾炎、全身性红斑狼疮相关血管炎、类风湿性关节炎相关血管炎、免疫复合血管炎、高安氏病、扩张型心肌病、糖尿病血管病变、川崎病、静脉空气栓塞,支架置入、冠状动脉旋磨术、经皮腔内冠状动脉成形术后再狭窄,重症肌无力、冷凝集素病、皮肌炎、阵发性寒冷性血红蛋白尿、抗磷脂综合征、格雷夫斯病、动脉粥样硬化、阿尔茨海默氏病、全身性炎性反应败血症、败血性休克、脊髓损伤、肾小球肾炎、移植排斥、桥本甲状腺炎、I型糖尿病、银屑病、天疱疮、自身免疫性溶血性贫血、特发性血小板减少性紫癜、古德帕斯彻综合征、迪戈斯病或灾难性抗磷脂综合征。
优选地,所述的药物的适应症为PNH。
又一方面,本发明提供了包括前述的抗C5抗体或抗体制剂的药物。
优选地所述的药物用于治疗PNH。
优选地,所述的药物的剂型为注射剂。
又一方面,本发明提供了前述的缓冲体系在抗体药物保存中的应用。
优选地,所述的抗体药物为抗C5抗体相关药物,如前述的抗C5抗体的保存。
本发明的有益效果:
本发明相对于现有技术改进了抗C5抗体的稳定性,改进了抗体制剂的相关缓冲体系,解决了抗体制剂的高粘度、聚集和电荷异质性改变等问题,提高了相应抗体高浓度液体制剂的稳定性。常规抗体制剂浓度一般在100mg/mL以下,本发明缓冲液体系抗体浓度达到200mg/mL左右。且本发明的抗体制剂表现出了良好的长期稳定性、亲和力和生物利用度。
附图说明
图1为不同浓度EA5抗体对补体依赖的细胞毒作用(CDC)的抑制活性。
图2为ICR小鼠单次静脉注射和皮下注射30mg/kg剂量EA5抗体药时曲线图。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1一种抗C5抗体制剂的制备方法
(1)抗体的制备
一种高稳定性的抗C5抗体,其重链序列为SEQ ID NO.2:轻链序列为SEQ ID NO.1。对其进行长效改造以增长半衰期,改造后抗体重链序列为SEQ ID NO.3;轻链序列为SEQ IDNO.1。
本实施例抗体的制备方法为本领域常规制备方法,通过构建表达载体,以稳转的方式转入宿主细胞CHO构建高表达稳定细胞株,对种子细胞进行批次补料培养,表达抗C5抗体,收获后经过亲和层析和离子交换层析后,得到抗C5抗体。
(2)缓冲液的制备
按照特定配方配制缓冲液,调节缓冲液pH至6.3。
(3)抗体制剂的制备
以180mg/mL抗体的浓度使用步骤(1)的改造后抗体和步骤(2)的缓冲液制备高浓度抗体制剂。制备过程如下:
将离心机转速设置为3500 rcf,蛋白用超滤离心管浓缩换液至制剂处方中,共换液155倍,调整蛋白浓度至目标浓度180 mg/mL,然后于超净台中将各处方样品过滤分装至2R西林瓶及5 mL储液袋中,西林瓶加塞轧盖。
处方1缓冲液:50 mM组氨酸-盐酸组氨酸、80 mM盐酸精氨酸,25 mM氯化钠,0.03%(w/v)PS80。调节缓冲液pH至6.3。
处方2缓冲液:20 mM组氨酸-盐酸组氨酸、80 mM盐酸精氨酸、25 mM氯化钠、0.03%(w/v)PS80。
处方3缓冲液:20 mM组氨酸-盐酸组氨酸、110 mM盐酸精氨酸、25 mM氯化钠、0.03%(w/v)PS80。
处方4缓冲液:20mM枸橼酸-枸橼酸钠+70mM 盐酸精氨酸+50mM氯化钠+0.03%PS80pH6.3。
实验例1抗体制剂质量属性研究
以处方1为例,对抗体制剂进行外观、蛋白浓度、渗透压、粘度、SEC-HPLC检测、CEX-HPLC、nrCE-SDS、结合活性、与C5亲和力及CDC效应测定。
(1)蛋白浓度的检测方法为:蛋白含量使用紫外分光光度计(岛津,型号:UV-1900)来检测。将蛋白消光系数为1.531(mg/mL)-1·cm-1。用超纯水清洗微量比色皿2-3次,分别在两个微量比色皿中加入超纯水,200µL/皿,做空白校正,再加入200 µL/皿样品,连续读取三次。通过消光系数、稀释倍数及OD值计算出样品的浓度。
(2)渗透压的检测方法为:将样品放置于渗透压仪测样探头上,仪器自动开始测量,1分钟后显示并打印测试结果。
(3)粘度的检测方法为:使用microVISC pipettes注射器吸取一定体积的样品,将注射器针头顶住进样口,上样完成后,仪器自动开始测量。
(4)SEC-HPLC的检测色谱柱厂家:Agilent,型号AdvanceBio SEC 300A,规格2.7µm。
高效液相HPLC厂家:Thermo,UltiMate 3000。
流动相组成为20mM PB+200 mM NaCl+100mM Arg,pH6.8。
采用面积归一化法积分,计算主峰,聚合体峰和单体后的片段峰。
SEC-HPLC纯度检测条件具体如下:
(5)CEX-HPLC纯度
色谱柱:厂家Sepax,型号proteomix SCX-NP5,规格4.6×250mm;高效液相色谱仪HPLC:厂家Thermo,型号UltiMate 3000。流动相A:20mM MES,pH 6.5;流动相B:20mM MES+500mM NaCl,pH 6.5。采用峰面积归一化对结果进行定量分析,计算酸性组分含量(主峰前所有峰面积百分比之和),主成分含量(主峰峰面积百分比)和碱性组分含量(主峰后所有峰面积百分比之和)。详细见下表:
CEX-HPLC纯度检测方法
(6)nrCE-SDS纯度
毛细管电泳仪:厂家SCIEX,型号PA800 Plus。毛细管电泳色谱仪:厂家G7100A,型号Aglient。先将样品脱盐,加pH6.5柠檬酸-磷酸盐缓冲液于离心浓缩中至400µL,13000rpm离心15min,将离心后的离心浓缩柱反插到新的离心管中,6000rpm离心3min后回收蛋白,加入250mM NEM溶液,混匀后70℃加热10min,冷却后10000rpm离心3min,即可进样。采用PDA检测器,检测窗口为100*800µm。从毛细管左端进样,毛细管的有效分离长度为20cm。采用电压进样,5kV持续20秒。采用恒电压分离,分离电压15kV。碱洗冲洗液正向70psi冲洗3min,酸性冲洗液正向70psi冲洗1min, 纯水正向70psi冲洗1分钟,SDS凝胶缓冲液正向70psi冲洗10min。采用峰面积归一化法,计算高分子组分含量(主峰前所有峰面积百分比之和),主成分含量(主峰峰面积百分比)和低分子物组分含量(主峰后所有峰面积百分比之和)。
(7)结合活性检测方法(ELISA):
用1 ×PBS包被缓冲液将250 µg/mL的C5抗原(购自Acro Bio,货号为CO5-H52Ha)稀释至0.5 µg/mL,100 μL/孔包被96孔酶标4℃过夜。洗板4次,250 μL/孔加入封闭液,室温孵育2小时。洗板4次,将梯度稀释好的样品100 μL/孔转移至96孔酶标板中,室温孵育1 h。洗板4次,100 µL/孔加入二抗工作液(1:20000稀释),室温静置孵育1 h。洗板4次,100 µL/孔加入TMB显色液,室温反应10 min,加入终止液。在酶标仪上以630 nm为参比波长读取450nm处吸光度值。以浓度为横坐标,OD450-630nm为纵坐标,做四参数曲线拟合,可直接得到EC50和R2,计算得到待测样品的相对结合活性。相对结合活性=参照品(或工作参比品)EC50/待测样品EC50×100%。
检测主要质量属性如下:
实验例2抗体制剂亲和力检测
(1)检测抗C5抗体与C5的亲和力,检测方法如下:
将带有生物素标记的C5用SD缓冲液(0.1% BSA,0.01% Tween 20,1×PBS)稀释至5μg/mL,再将供试品稀释至50μg/mL,平行制备三份,将稀释好的供试品、FcRn和SD缓冲液分别加入黑色96孔板中,将SA Sensor和黑色96孔板放入设备仪器中,设置好程序进行曲线拟合和数据分析。以处方1所示制剂检测示例结果如下:
结果表明,本发明的抗C5抗体与C5具有较好的亲和力。
(2)检测抗体与FcRn的亲和力,检测方法如下:
实验前将SD缓冲液pH调节至6.0,将带有生物素标记FcRn用SD缓冲液稀释至5μg/mL,再将供试品稀释至50μg/mL,平行制备三份,将稀释好的供试品、FcRn和缓冲液分别加入黑色96孔板中,将SA Sensor和黑色96孔板放入设备仪器中,设置好程序进行曲线拟合和数据分析。以Eculizumab(参见US 6355245,构建表达质粒,采用293T细胞加PEIUS5736137A瞬时转染表达,并经一步亲和层析后加入缓冲液后保存使用)为阳性对照。
检测结果如下:
检测结果表明,本发明的抗体制剂中的抗体与FcRn的结合能力优于阳性对照。
(3)检测不同浓度抗体的CDC效应。检测方法为:
取适量的靶细胞(人Burkitt's淋巴瘤细胞,ATCC CCL-213)加入10 μg/mLRituximab(参见US5736137A,构建表达质粒,采用293t细胞加PEI 瞬时转染表达,并经一步亲和层析后加入缓冲液后保存使用)孵育半小时,再加入浓度范围216ng/mL-12500ng/mL的抗体药物,孵育3小时,取出平衡至室温,加入Cell titer检测试剂(Promega,货号G7572),避光震荡后读数。以抗体药物浓度为横坐标,RLU值为纵坐标进行四参数曲线拟合,其中RLU值与细胞裂解率呈正相关。结果见图1。图1表明随着抗体浓度的变低,细胞裂解率增加,Rituximab引起的补体诱导CDC效应增强,而高浓度的抗C5抗体可以抑制Rituximab引起CDC效应。
实验例3加速稳定性实验
处方1-4抗体制剂于40℃条件下放置14天,并参照实验例1检测其起始参数(T0)和14天参数以评估制剂稳定性,结果如下:
实验例4皮下注射生物利用度
以处方1为例,抗体制剂进行生物利用度研究,具体方法为:本实验通过给CD-1小鼠皮下和静脉注射抗体制剂后,观察抗体在小鼠体内血药浓度经时变化情况,获得药物的基本药代动力学参数,了解受试物的基本动力学特征。
具体方法如下:
根据体重随机分组,每组6只,雌雄各半。使用ICR小鼠(8周龄,体重30g)进行单次尾静脉注射或单次皮下颈部注射抗体制剂,给药量10mL/kg。
采样时间:给药结束即刻(1 min内),给药后1 h±5 min、6 h±5 min、24 h±5min、48 h±5 min、72 h±5 min、96 h±5 min、120 h±5 min、168 h±5 min、240 h±5min、336 h±5 min、504 h±5 min和672 h±5 min;采血量:约0.2 mL/只/次;采集动物:3只/时间点/性别/组;采血方式:眼眶静脉。血浆样本中抗体的浓度使用经方法学验证的ELISA分析方法进行分析检测。所有样品均复孔测定。血浆中抗体的浓度和时间曲线通过非房室模型进行分析。
ELISA法测定血浆中抗体的含量:
将山羊抗人的多抗(购自Abcam,货号为Ab6858)包被在疏水酶标板上,用双抗夹心法测定血浆中EA5含量。结果见图2,图2表明了:相比静脉注射,皮下注射后主要经淋巴***吸收,药物达峰时间在24小时,平均Cmax在240μg/mL,生物利用度为70%左右。
Claims (9)
1.一种抗体制剂,其特征在于,由抗C5抗体和缓冲体系组成;
所述的抗C5抗体轻链序列为SEQ ID NO.1,重链序列为SEQ ID NO.3;
缓冲体系由20-50mM组氨酸-盐酸组氨酸、70-110mM精氨酸、25-50mM氯化钠和0.03%PS80组成;
所述的抗C5抗体含量为160-200mg/mL。
2.根据权利要求1所述的抗体制剂,其特征在于,缓冲体系由20mM组氨酸-盐酸组氨酸、70-110mM精氨酸、25-50mM氯化钠和0.03% PS80组成。
3.根据权利要求2所述的抗体制剂,其特征在于,缓冲体系由20mM组氨酸-盐酸组氨酸、80mM精氨酸、25-50mM氯化钠和0.03% PS80组成。
4.根据权利要求3所述的抗体制剂,其特征在于,缓冲体系由20mM组氨酸-盐酸组氨酸、80mM精氨酸、50mM氯化钠和0.03% PS80组成。
5.根据权利要求4所述的抗体制剂,其特征在于,缓冲体系pH为6.0-6.5。
6.根据权利要求5所述的抗体制剂,其特征在于,缓冲体系pH为6.0-6.3。
7.权利要求1-6任一项所述的抗体制剂在制备药物中的应用,其特征在于,所述的药物用于治疗类风湿性关节炎、抗磷脂抗体综合征、狼疮性肾炎、缺血再灌注损伤、非典型溶血性***综合征、典型溶血性***综合征、PNH、致密物沉积病、视神经脊髓炎、多灶性运动神经病、多发性硬化、黄斑变性、HELLP综合征、自发性流产、血栓性血小板减少性紫癜、寡免疫性血管炎、大疱性表皮松解症、复发性流产、创伤性脑损伤、心肌炎、脑血管病症、周围血管病症、肾血管病症、肠系膜/肠血管病症、血管炎、过敏性紫癜性肾炎、全身性红斑狼疮相关血管炎、类风湿性关节炎相关血管炎、免疫复合血管炎、高安氏病、扩张型心肌病、糖尿病血管病变、川崎病、静脉空气栓塞,支架置入、冠状动脉旋磨术、经皮腔内冠状动脉成形术后再狭窄,重症肌无力、冷凝集素病、皮肌炎、阵发性寒冷性血红蛋白尿、抗磷脂综合征、格雷夫斯病、动脉粥样硬化、阿尔茨海默氏病、全身性炎性反应败血症、败血性休克、脊髓损伤、肾小球肾炎、移植排斥、桥本甲状腺炎、I型糖尿病、银屑病、天疱疮、自身免疫性溶血性贫血、特发性血小板减少性紫癜、古德帕斯彻综合征、迪戈斯病或灾难性抗磷脂综合征中的一种或多种。
8.根据权利要求7所述的应用,其特征在于,为皮下注射药物。
9.包括权利要求1-6任一项所述的抗体制剂的皮下注射药物。
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