CN110612153A - 纯化透明质酸的方法 - Google Patents
纯化透明质酸的方法 Download PDFInfo
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- CN110612153A CN110612153A CN201880030814.6A CN201880030814A CN110612153A CN 110612153 A CN110612153 A CN 110612153A CN 201880030814 A CN201880030814 A CN 201880030814A CN 110612153 A CN110612153 A CN 110612153A
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- streptococcus
- purified
- bacillus
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Classifications
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Abstract
描述了一种纯化HA的方法,以及包含由此纯化的HA的药物、化妆品和营养组合物。
Description
发明领域
透明质酸(HA)是一种线性、阴离子型且不含硫酸基团的高分子量多糖,由D-葡萄糖醛酸和N-乙酰基-D-葡萄糖胺的交替残基组成。它天然存在于细胞周围的凝胶中,存在于脊椎动物生物体的结缔组织的基本物质中(其代表主要成分之一),存在于关节的滑液中,存在于玻璃体液中和存在于脐带中。因此,HA在生物有机体中起着重要作用,首先是对许多组织(如皮肤、肌腱、肌肉和软骨)的细胞提供机械支持。
还已知HA通过其膜受体,特别是CD44、CD54和CD168,调节与细胞的生理和生物学有关的许多不同过程,例如增殖、迁移、细胞分化和血管生成,以及它还执行其他功能,例如组织水合作用和关节润滑。它具有绝对的生物相容性,并且由于其许多特殊功能,多年来已广泛用于从组织修复到粘液补充疗法,从皮肤美容药物到眼内手术,从组织工程到细胞治疗等各个领域。
HA的化学、物理和生物学特性与它的分子量(MW–指通过“固有粘度”方法计算的重均分子量)密切相关,这是非常可变的:通常可以说,HA的重均分子量大约从20,000到13x106Da变化,并且所述大约是必须的,因为它会根据来源以及用于分离它的生产和纯化方法而发生根本变化。
基本上有两种获取HA的基本方法:
从动物来源生产:从历史上讲,HA是从动物组织(例如脐带、玻璃体液或牛滑液)中提取的,尤其是公鸡冠。动物来源的生产有很多限制,价格昂贵,例如,由于需要大量步骤来去除各种杂质(从消化起始组织后的大量有机残留物开始),因此需要采取一些步骤来确保灭活和去除可能存在于起始原料中的任何污染剂(例如病毒),它需要大量的原料,并且收率不高;
微生物的发酵:某些微生物,特别是链球菌(Streptococcus)或巴斯德氏菌(Pasteurella)属的微生物,如果经过适当的刺激和/或修饰,能够产生HA,HA分泌在培养液中,通过对本领域技术人员已知的不同方法从中分离。同样在这种情况下,需要许多步骤来去除存在的“杂质”,例如,所用微生物的细胞壁残留物、金属离子、核酸和任何其他不希望的蛋白质物质。尽管有这些限制,但这仍然是生产HA的最成熟和使用最广泛的方法。
通过生物技术生产HA的新方法也在研究中,通过在合适的宿主细胞,例如芽孢杆菌属(Megaterium和Subtilis)和大肠杆菌中转染表达HA合酶的基因。然而,对于这些生产方法,去除所有潜在有害残留物所需的所有程序也是必需的。
无论如何,无论采用哪种方法,获得HA的关键步骤显然是多糖的提取和纯化阶段。有许多已知的方法,所有这些方法都非常明确,并且相对于获取HA的起始来源而言显然是经过调制的。
首先,必须去除来源的残留物,因此,为了从动物组织中提取蛋白质,存在蛋白质的消化阶段,然后进行过滤,离心和洗涤。对于发酵,通常进行离心和逐步洗涤。在任何情况下,都会获得液体级分,然后从中分离出多糖。在这方面,最广为人知且肯定是最常用的方法,尤其是对于动物来源的HA,是溶剂沉淀法:对于较大的生产线,在上述液体级分上使用增加浓度的有机溶剂(乙醇、丙酮),引起透明质酸沉淀,然后将其通过随后的溶解和沉淀进行纯化。
另一种系统涉及使用季盐,例如鲸蜡基吡啶鎓或鲸蜡基三甲基铵,其具有使多糖络合并诱导其沉淀的功能。同样,随后的溶解和沉淀对于获得最终产物是必需的。
技术的发展还结合了上述关键步骤,以使该方法在收率方面有效且在纯度方面有效:然而,迄今为止,每年仍有几百种数量级的不良事件向主管机构(例如FDA)报告,特别是在注射了基于HA的药物组合物之后。
透明质酸被广泛用于各种领域和病理学:从具有保湿作用的化妆品(局部或口服)到具有持久作用的局部皮肤美容,从用于皮肤缺陷(皱纹或疤痕)(皮肤内)的矫正的注射装置,到更严格的药理学应用(例如在骨关节疾病中的关节内使用),眼内替代玻璃体液,在膀胱内用于治疗间质性膀胱炎等。
对于不接触受损组织的化妆品应用而言,化妆品级HA(纯度较低)就足够了,很明显,在制药应用中,尤其是可注射应用,甚至在封闭腔(关节和眼睛)中也是如此,需要一定程度的绝对纯度:由于从中提取透明质酸的材料的性质,实际上,最终产品中可能存在残留形式的核酸,蛋白质和/或细胞壁的残留细菌性毒素,革兰氏阳性菌(例如芽孢杆菌属,链球菌,肠球菌和葡萄球菌),例如脂蛋白酸LTA或革兰氏阴性菌(例如埃希氏大肠杆菌,巴斯德氏菌和沙门氏菌),例如脂多糖LPS。这些各种各样的污染物能够引起明显的炎症反应,从而在局部和全身水平释放细胞因子(尤其是TNF和IL-1),进而引起全身性炎症反应,并在体内产生反应。整个生物体,在最严重的情况下,会导致败血性休克。
LTA和LPS实际上是由脂类部分和糖类部分组成的聚合物,能够引起强烈的免疫反应,在最严重的情况下,会引起关节炎,肾炎,脑膜炎或引起发烧和休克,其后果也可能致命。这解释了如上所述报告的大量不良事件。
除此之外,还应考虑到,如前所述,HA的分子量相对于来源和生产方法是可变的,并决定了其应用领域:例如,低分子量用于皮肤病学或皮肤美容制剂(约200kDa,),而对于关节内应用,较高的MWs是优选的(通常为700至1,800kDa; ),达到的MWs高于1,500kDa,用于美容手术或眼内手术。但是,在纯化过程中,必须去除分子量低于30,000Da的HA级分,因为该级分已被广泛证明具有很强的炎症作用(EP0138572),无论哪种应用,这都是绝对不希望的。
这意味着在HA的工业纯化过程中,必须评估和控制各种因素:
工艺收率:从所选的生产来源中提取最大可能量的HA是关键的;
工业便利性:必须以浪费最少的所用材料(试剂,溶剂等)获得最佳产品,产生尽可能少的处理残留物并且时间要尽可能短;
纯度:获得的产物必须不含任何污染物,并且也必须不含已知能够引发炎症级联反应的MW<30,000Da的HA级分。
纯度显然与纯化步骤的准确性有关。
针对总结的这些要求的许多尝试在现有技术中是已知的。在众多尝试中,可提及以下:
EP0138572:使用,尤其是超滤步骤,添加季盐(鲸蜡基吡啶鎓)和化石树脂,用乙醇沉淀,从公鸡冠中纯化HA,并获得两个MW级分(50-100kDa和500-730kDa),不含炎性部分;在这种情况下,超滤用于清除所有分子量小于30,000的炎性分子,并用于分离出两个所需的HA级分。
EP535200:通过用季胺皂化并随后用溶剂(乙醇或丙酮)沉淀从公鸡冠中纯化HA。获得的HA具有750至1,230kDa的可变MW,无炎性级分,特别适合眼科使用。
US6489467:通过用HCl加强酸化,随后改变pH和透析,从链球菌(Streptococcus)中纯化HA,获得MWs约为1,700kDa的HA;
Choi等人,生物材料研究,2014,18,1-10:通过超滤和丙酮沉淀从兽疫链球菌(Streptococcus zooepidemicus)中纯化HA。获得的HA的MWs范围为900至1100kDa;
EP2870255:通过过滤(去除杂质),超滤(将产物浓缩在其所存在的溶液中中),pH值变化以及最后用乙醇沉淀从兽疫链球菌(Streptococcus zooepidemicus)中纯化HA,得到的MW范围为60至2,400kDa;
EP1543103:从链球菌(Streptococcus)培养物中纯化HA,通过在先前过滤的培养液中添加芳香族树脂,以吸收大部分杂质,然后进行超滤以浓缩HA溶液,最后用乙醇沉淀。
WO2018/020458:从链球菌(Streptococcus)属或芽孢杆菌(Bacillus)属的微生物培养物中纯化HA,通过热处理分离成具有精确分子量(92-230kDa;450-780kDa;920-1450kDa)的级分。除其他阶段外,纯化包括在芳族树脂中的步骤,然后重复过滤,最后用有机溶剂沉淀并进行相对洗涤。
尽管本文引用的方法和通常使用的常规方法能够产生高质量的透明质酸并具有可接受的收率,但它们极其复杂,因此在使用的试剂、溶剂、过滤器等方面非常昂贵,在时间上,以及最终在去除加工残留物所需的成本方面也非常昂贵。
本发明通过极其简化的透明质酸钠盐的纯化方法克服了现有技术的缺点,其允许获得非常高纯度的产物,同时惊人地节省了材料和时间,并且显著提高了迄今为止的工业收率,其值非常接近100%。
具体实施方式
本发明的一个目的涉及一种提取透明质酸并随后以碱金属和/或碱土金属盐的形式,优选以钠盐形式的纯化的新方法,其特征在于:
很高的收率;
工业便利,因为去除了许多中间步骤;
非常高的最终产品的纯度,完全没有任何污染物。
本申请人开发的透明质酸的提取方法极为高效低耗,因为它提供了严格限制的操作步骤以达到最终产品。这不仅意味着节省了所用材料(溶剂、试剂、盐、化石树脂、合成树脂、过滤器等),而且在涉及加工残留物的处理方面也节省了:众所周知,化学工业中所使用的材料必须按照安全程序进行处理,例如考虑有机溶剂的情况。最后,在这种情况下,较少的步骤数也相应地减少了加工处理时间。这些因素的结合带来了更好的工业便利性。该方法可以用于纯化根据本领域技术人员已知的众多技术中的任何一种制备的HA:HA实际上可以源自生物来源,特别是来源于鸡属的鸡冠(EP0138572),来源于链球菌的发酵;来源于枯草芽孢杆菌和巨大芽孢杆菌的分子工程(EP2614088,EP2614087);该方法优选适用于从链球菌,特别是马链球菌马亚种,68222,突变体H-1发酵得到的HA(EP0716688)。
如申请人进一步证明的,本申请所要求保护的方法使得可以制备极高纯度的HA,其不仅符合欧洲药典(Ph.Eur.5.0 1472)要求的所有化学/物理规格,而且甚至更高,尤其在细菌内毒素、蛋白质、热原的含量方面。
应当记住,实际上,无论生产来源如何,如果最终产品没有经过完美的纯化,它都可能会包含各种污染物,例如热原,蛋白质(源自起始的生物材料),核酸,源自革兰氏阳性细菌(链球菌或芽孢杆菌,或肠球菌和葡萄球菌),或革兰氏阴性细菌(例如大肠杆菌,巴斯德氏菌和沙门氏菌)的细菌来源的毒素:在透明质酸终产物中存在这些毒素,例如脂二十二酸(LTA)或脂多糖(LPS),将会由于产生高度促炎因子的高风险而无法使用,从而导致治疗的关节或组织炎症和/或感染,在最严重的情况下,会导致其完全破坏或坏死。
最后,根据本发明的纯化方法的收率极高,稳定在85%至100%的范围。
根据本发明获得的透明质酸可以完全安全地使用,尤其是在所有注射药物组合物中(关节内、皮内和眼内),因为它不含任何促炎和热原性成分。用所述新方法纯化的HA,本发明的目的,优选以碱金属或碱土金属盐的形式获得,甚至更优选以钠盐的形式获得,也可以用于制备本领域技术人员已知的所有衍生物,例如HA与重金属的盐(EP0827514)、酯(EP0216453)、酰胺(EP1095064)、硫酸化产物(EP0940410)和包括HA的自交联产物的交联产物(EP0341745)。
本发明的另一个目的涉及含有通过本发明的纯化方法获得的HA的药物、化妆品和营养组合物,特别是:
-用于关节炎关节粘度补充、外伤性关节损伤、软骨下损伤的关节内用药物组合物;
-用于眼内使用或眼部给药的药物组合物,用于治疗眼疾;
-用于预防术后粘连的药物组合物;
-用于局部和注射的药物组合物(皮内和/或肌肉内),用于皮肤溃疡、褥疮、各种程度的烧伤、疤痕和皮肤损伤的治疗中,用于瘢痕疙瘩或增生性/肥大性疤痕的治疗中,用于各种类型的完整或受损皮肤的皮肤缺陷的治疗中,以及作为皮肤病的治疗疗法,例如湿疹和各种皮炎,特别是特应性和尿布性皮炎,牛皮癣;
-用于膀胱内的药物组合物,特别是用于治疗间质性膀胱炎;
-注射用药物组合物,在皮肤美学中用作填充剂或在美容手术中用作塑形;
-用于局部和口服的化妆品组合物;
-用于关节炎关节的口服治疗、肌腱营养、皮肤营养和胃肠道粘膜营养的药物或营养组合物。
本发明的另一个目的涉及二维/三维生物材料,其包括用根据本发明纯化的HA制备的衍生物,其形式为垫,织物,无纺布,颗粒,薄膜和凝胶,还可以与各种来源的细胞和/或血液衍生物,例如血小板衍生物结合。
如已经指出的,根据本发明的方法可以应用于根据各种已知技术获得的HA的纯化,因为通常,所有方法都提供了一些通用步骤,可以概括如下:
生产:
根据选择的来源,进行生物材料的消化,或适当确认的微生物的发酵,或再有,根据新的领域,采用生物技术进行生产。所获得的基本上是被固体生物材料(所谓的生物质)严重污染的发肉汤,其在溶液中含有透明质酸和许多其他完全不希望的各种类型的物质。每种方法还提供了去除生产残留物的步骤(包括固体和尽可能溶解的),通常是通过使用各种方法分离生物质,从而得到含有HA的过滤肉汤,进行后续提取步骤:
在这一步骤中,从发酵肉汤中分离并回收HA。正是在这一步骤中,各种已知方法在操作方法(试剂,溶剂,过滤器,表面活性剂等)方面以及步骤数量上以及因此在时间方面具有最大不同。当然,这是该过程中最复杂,最精细的部分,因为正是在萃取步骤中,才为最终产品的纯度和工业过程的收率奠定了基础。提取步骤的结果通常是HA的浓缩溶液,然后将其进行
所谓纯化步骤:
尽管还根据透明质酸的来源对纯化方法进行了校准,但通常,它们遵循本领域技术人员已知的共同原则:
处理溶液中含有透明质酸的液相,以去除可能存在的有害成分(蛋白质,毒素等);
然后用HA不溶于其中的有机溶剂(如乙醇,丙酮或其混合物)进行沉淀。
然后将沉淀物反复溶解在水性介质中,随后进行沉淀,溶解,过滤和洗涤,直至获得最终产物,然后将其干燥。
根据本发明,广义上的纯化过程(理解为从发酵肉汤开始直至最终产物的步骤的组合),无论HA的来源如何,在迄今已知的各种方法中都聚焦于提取步骤,其在许多段落中都有阐述。
实际上,在此步骤中,必须从肉汤中分离出尽可能最大量的所需产物(HA),同时除去尽可能最大量的各种杂质,这解释了为何需要大量的操作步骤。
因此,正是在此步骤中,定义了要评估纯化过程的基本参数,即:
收率,即获得的产品的量;
所得产品的纯度;
工业便利性,即材料的浪费和获得具有所需特性的所需产品的时间。
申请人惊奇地发现,从来自生产步骤的肉汤(无论可能是什么)开始,经过减少的一系列步骤,根据本发明的提取方法实际上使得存在于肉汤中的所有透明质酸都可以被从中提取出来,并且所述HA,在根据已知技术的适当纯化步骤之后,被证明在热原、蛋白质、细菌内毒素方面是极其纯净的。
因此,本发明的一个目的涉及从发酵肉汤,优选从链球菌或芽孢杆菌属的微生物,特别是马链球菌、枯草芽孢杆菌或巨大芽孢杆菌,更优选为马链球菌的微生物的发酵肉汤中提取和纯化HA的方法,所述方法的特征在于,其包括萃取步骤,该萃取步骤包括以下步骤或由以下步骤组成:
a.将过滤的肉汤用纯净水稀释至相对于初始体积的1.1至3体积,优选等于1.5的体积;
b.在包含超滤膜的切向流过滤器(TFF)盒内强制再循环通过合并回流液和透过液形成的来自步骤a.的肉汤,所述超滤膜由芳基磺酸聚合物材料,优选聚醚砜,制备,孔隙率范围为5,000至300,000道尔顿,优选50,000至200,000,甚至更优选等于100,000道尔顿,其中重复进行所述强制再循环1-6小时,优选等于3小时,所述再循环以单向流在密闭系统中以恒定体积进行,没有从外部引入液体。
本发明的另一个目的涉及从发酵肉汤,优选从链球菌属或芽孢杆菌属的微生物,特别是马链球菌、枯草芽孢杆菌或巨大芽孢杆菌,更优选地马链球菌的微生物的发酵肉汤中提取HA的方法,所述方法包括以下步骤或由以下步骤组成:
a.用纯净水将过滤后的发酵肉汤稀释为初始体积的1.1至3倍,优选等于1.5倍;
b.在包含超滤膜的切向流过滤器(TFF)盒内强制再循环通过合并回流液和透过液形成的来自步骤a.的肉汤,所述超滤膜由芳基磺酸聚合物材料,优选聚醚砜,制备,孔隙率范围为5,000至300,000道尔顿,优选50,000至200,000,甚至更优选等于100,000道尔顿,其中重复进行所述强制再循环1-6小时,优选3小时,所述再循环以单向流在密闭系统中以恒定体积进行,没有从外部引入液体。
本申请人开发的提取方法使得可以几乎完全提取,因此可以回收最初过滤的肉汤中存在的透明质酸,达到的收率范围为95%至100%。
因此,如此高的提取收率导致成品收率比目前已知的要高得多,因为在这些过程的典型后续沉淀和洗涤步骤中,HA的定量损失仅是与操作有关的损失(例如,最少的HA附着在所使用的设备上);因此,认为提取步骤的收率可以强烈预测整个纯化过程的收率。
根据本发明的HA的提取和纯化方法可以提供在强制再循环步骤
b.之后进行以下步骤:
c.用选自纯水和盐水溶液的透析溶液,优选纯水进行包含在步骤b.的TFF盒中回流液的透析I;
d.浓缩从步骤c得到的体积至起始肉汤的体积;
e.用选自纯水和盐水溶液的透析溶液,优选纯水进行从步骤d.得到的体积的透析II,重复所述透析5至15次,优选6-12次;
f.最终浓缩从步骤e得到的体积,直到获得的最终体积等于起始肉汤的三分之一;
g.通过纯水的循环进行保留在TFF盒内的产品的回收,直到获得等于起始肉汤的一半的总体积。
根据本发明的提取方法在超滤技术(UF)的创新使用中具有其突出意义。在一般情况下,UF技术是已知的方法,其允许从包含所述组分的液相中分离出组分,并且其分子量高于由过滤膜的孔(截止)限定的分子量。非常示意性地,包含在特定储罐中的液相(进料)被泵推向带有细孔(截止)的过滤膜上。穿过滤膜的流体在下游收集,形成滤液或透过液,然后进行处理。待回收的组分保留在膜的表面上,形成含有目标产物的回流液。为了回收尽可能多的产品,将回流液进行多次UF循环,将其重新引入到储罐中,然后将其作为进料重定向至UF盒,同时去除每次获得的透过液。透过液的逐渐去除显然由于体积损失而导致浓缩,然后通过向罐中添加合适的液体进行补偿。透过液的逐渐去除显然导致透明质酸的损失,这是UF过滤器无法完全保留的。
为了获得尽可能纯的最终产品,如本领域技术人员所知,在循环之后进行一个或多个透析步骤,从外部引入合适的液体(透析溶液),以便产生清洗流,该清洗流在流过回流液时有助于去除仍然存在的杂质。当如本发明的情况那样,当必须分离生物学来源的分子时,通常在分子量方面具有显著的尺寸,则采用的UF是所谓的切向流过滤(TFF)。
因此,申请人开发了一种用于从发酵肉汤中提取HA,优选从链球菌或芽孢杆菌属的微生物,特别是马链球菌、枯草芽孢杆菌或巨大芽孢杆菌,更优选地从马链球菌的发酵肉汤中提取HA的创新方法,一种工艺,其通过创新地使用TFF技术,使得透明质酸从包含它的肉汤中几乎完全回收。
申请人实际上已经令人惊讶地发现,通过在TFF之前进行稀释步骤并随后对处理的培养肉汤进行强制再循环,实际上可以完全回收存在于该同一培养肉汤中的透明质酸。如下所详细描述,通过将培养肉汤送到适当的TFF盒中,收集所获得的透过液(没有如现有技术的要求去除透过液),并将其输送到原始肉汤所用的储罐中后,再次将其重定向到相同的TFF盒,在其中将作为进料使用:通过这种方式,仍存在于再循环进料中的HA将构成进一步的回流液,并且逐渐产生的透过液将重复上述循环(强制再循环)。因此,在该步骤中,回流液和透过液在包含初始进料的罐中合并,并以单向流再循环。所有这些都是在不从外部引入液体的情况下进行的,因此在系统内部(因此在封闭系统中)保持体积恒定。整个系统自然配备了一系列调节流量和流出的阀,用于输送液体的可变压力泵,用于控制压力值的压力表。
如上所述,所述的这组操作确定了从起始肉汤中几乎全部回收HA,收率为95%至100%。
因此,很明显,本发明实质上改变了现有技术水平,显著提高了所述方法的收率(更多的透明质酸被回收),并且在工业上具有一系列的便利性(较少的步骤意味着使用更少量的材料,过滤器等...以及待处理的废物的量更少)。
更具体地,如已经指出的那样,进行本发明所述方法的操作流程从含有HA的肉汤开始并且来自任何初始生产步骤,优选来自链球菌的发酵,特别是来自马链球菌马亚种,68222,突变体H-1的发酵(EP0716688)。在这种特定情况下,用强酸,优选HCl的酸溶液将肉汤的pH调节至4.0至5.0,优选4.5,随后通过本领域技术人员已知的分离技术(离心和/或微滤和/或在化石粉垫上的过滤)与生物质分离。
过滤后的肉汤的体积显然根据工厂的生产能力而变化;在这种特定情况下,容量可以从2,000升到4,000升不等;该方法优选用于3,000升的批量。接下来是提取步骤:
将如此获得的肉汤依次进行以下步骤:
a.稀释:将来自生产步骤并如上所述处理的肉汤引入合适的罐中,并用纯净水稀释至与初始体积相比的总体积1.1至3体积,优选为1.5体积。这是一种完全创新的方法,因为根据现有技术,将起始肉汤是被浓缩的,从而使得必须处理的体积较小;
b.强制再循环:将如此稀释的肉汤(进料)通过包含泵的系统在含有UF膜的TFF盒内循环,所述UF膜由芳基磺酸聚合物材料(优选聚醚砜)制备,其孔隙率(截止值)为5,000至300,000道尔顿,优选50,000至200,000道尔顿,甚至更优选等于100,000道尔顿,不引入超滤溶液且不排出透过液,因此在密闭系统内。因此,产生了肉汤的强制再循环,其中透过液不是像通常那样被排除,而是被重新引入罐中,并与逐渐产生的回流液结合并进一步输送至滤膜。实际上,至少在第一循环中,透过液不仅包含无机盐和各种类型的杂质,而且还包含溶液中的HA。肉汤的强制再循环还会在UF盒内形成HA的凝胶状层,起着作为为仍存在于透过液中的HA的另一个过滤器的作用,同时防止系统堵塞。应该指出的是,强制再循环和整个萃取系统的特征在于液相(进料和其他液体)的单向流动。维持强制再循环的时间为1到6个小时,最好等于3个小时,并以30分钟的固定间隔进行测试,直到样品中的透过液与粉状NaCl混合,形成在NaCl中的0.3M最终摩尔浓度,并且添加两倍体积的乙醇不会产生任何沉淀(如前所述,用乙醇沉淀是从液相中分离HA的最简单和最直接的技术之一)。这意味着初始肉汤中存在的基本上所有HA都已保留在TFF盒中回流液中:显然,创新实际上去除了产品损失,从而带来了巨大的工业优势。
萃取步骤的收率用咔唑法(Ph.Eur.5.0;1472,01/2011)计算。简而言之,用咔唑法测定发酵结束时肉汤中存在的HA的浓度和提取步骤结束时获得的HA的浓度。更具体地,该比率是在根据描述提取的HA的克数与发酵结束时的初始肉汤的升数之间计算的;一个简单的比例就可以计算出收率值,表示为提取的HA与肉汤中初始HA的百分比。
因此计算出的在本发明中开发的萃取步骤的收率为95-100%。按照本领域技术人员所已知的进行后续步骤。特别地,申请人进行透析步骤,并且优选如下进行:
c.透析I:完全排空了HA含量的前一步骤b.产生的透过液通过打开排放阀去除。透析溶液的供应被打开并保持连续,从而通过回流液形成恒定的透析溶液流,就像通过去除逐渐形成的透过液一样,保持在罐内循环的体积不变。所用的透析溶液可以是纯净水或盐水溶液,优选纯净水。维持该程序必要的时间,从而使得去除的透过液的体积等于在强制再循环步骤结束时储罐中存在的透过液的体积的两倍。
d.浓缩:保持排放打开并阻断透析溶液的供应,将储罐中的内容物浓缩,直到使其回到初始稀释之前的起始液的体积(因此在步骤a.之前)。
e.透析II:如步骤c.中所述,对源自步骤d的体积重复透析步骤5-15次,优选6-12次。
最后,根据已知技术,优选通过最终浓缩进行HA的回收:
采取与步骤d.中所述相同的步骤,直到获得等于初始体积(在步骤a.之前)的三分之一的最终体积,
以及随后的回收:
通过循环纯净水回收保留在盒中的产品,直到获得的体积等于初始稀释之前的肉汤体积(因此在步骤a.之前)的一半。
此时,通过上述方法获得的溶液中存在的HA进入特定的纯化步骤,该纯化步骤可由本领域技术人员适当选择,因为该步骤是公知的技术,然后进入后续的干燥步骤。
简而言之,当应用于通过从链球菌属或芽孢杆菌属,特别是马链球菌的微生物发酵产生的HA的整体提取和纯化方法中时,本发明的目的涉及一种用于HA提取的在收率、产品纯度和工业便利性方面高效的方法。
其完全创新的特征在于,在TFF之前先进行稀释步骤和强制再循环被处理的培养肉汤;无论HA的来源、该来源的处理方式以及其到达干燥形式最终产品(准备好用于其最终用途)的方式,均可将该方法成功应用于HA的生产和纯化的任何已知全球流程中。
更具体地,申请人要求保护一种从根据已知技术获得的肉汤中以高收率提取HA的方法,所述肉汤特别是通过从链球菌发酵,更优选地通过马链球菌马亚种,68222,突变体H-1(EP0716688)的发酵而获得,包括以下步骤或由以下步骤组成:
萃取:
a.稀释:将过滤后的生产肉汤用纯净水稀释至相对于初始体积为1.1至3体积,优选等于1.5的体积;
b.强制再循环:来自步骤a的肉汤。在包含超滤膜的切向流过滤器(TFF)盒内强制再循环通过合并回流液和透过液形成的来自步骤a.的肉汤,所述超滤膜由芳基磺酸聚合物材料,优选聚醚砜制备,孔隙率范围为5,000至300,000道尔顿,优选50,000至200,000,并且甚至更优选等于100,000道尔顿。重复所述强制再循环1到6个小时,最好等于3个小时,直到HA完全保留在回流液中,所述再循环以单向流在密闭系统中以恒定体积进行,没有从外部引入液体。
然后采用根据本领域技术人员已知的方法,特别是透析步骤,优选如下
c.透析I:用选自纯净水和盐水溶液,优选纯净水的透析溶液进行透析,并保持去除等于前一步骤b.之后罐中存在的透过液体积两倍的透过液所需的时间;
d.浓缩:将罐中的内容物浓缩至起始肉汤的体积(在步骤a.之前);
e.透析II:对在步骤d.得到的体积重复根据步骤c.中所述的透析5-15次,优选6-12次。
f.最终浓缩:浓缩从步骤e.得到的体积,直至获得等于初始体积(在步骤a.之前)的三分之一的体积;
g.回收:保留在盒中的产品通过纯净水的循环进行回收,直到获得的总体积等于步骤a.之前肉汤的一半。
一旦完成了本文所述的所有步骤,就使用本领域技术人员已知的技术进行所谓的纯化步骤;仅以举例的方式,可以提及用强的碱金属或碱土金属碱,优选碱金属碱,尤其是NaOH处理,以去除任何其他可能的污染物,然后过滤(例如,在炭和/或过滤布和/或聚丙烯上),在有机溶剂(例如乙醇)中以不同的稀释度沉淀,最后在有机溶剂(优选乙醇)中洗涤;在各种纯化方法中,特别优选WO2018/020458的实施例3中所述的方法,该方法是本说明书的组成部分。
不管选择哪种纯化方法,都获得成盐的HA,优选以钠盐的形式获得成盐的HA。
然后将如此纯化的成盐的HA进行干燥,以使其最佳保存,然后再用于其最终用途。
由此获得的产物不仅证明符合欧洲药典的参数(Ph.Eur.5.0;1472),而且在细菌内毒素和蛋白质的含量方面甚至比其要求更高。
使用前面描述的咔唑方法(Ph.Eur.5.0;1472)计算得出的最终方法收率证实,在这一阶段,由于操作限制,产品损失最小。收率实际上落在85至100%的范围内,优选在95至100%的范围内。
实施例1:从马链球菌的发酵肉汤中提取HA
通过在搅拌下加入1N HCl溶液使3,000升马链球菌马亚种,68222,突变体H-1的的发酵肉汤的pH值达到4.5,然后通过硅藻土垫(硅藻土化石粉)过滤除去生物质。
然后将如此处理过的肉汤用1,500升纯净水稀释,直至最终体积为4,500升,并将其置于具有适当容量(6,000升)的反应器中。
使用配备有孔隙率(截留值)等于100,000道尔顿的聚醚砜膜的TFF盒(Pall,Omega单元),对获得的肉汤体积进行强制再循环步骤。在通过泵施加的入口压力不超过2.5巴的压力下将进料引入到料盒中。维持强制再循环3小时,并且每隔30分钟进行一次测定,以评估透过液中HA的存在:将32ml NaCl和两倍体积的乙醇添加到2ml透过液中。强制再循环3小时结束时,测试结果为阴性,即未形成沉淀,这意味着HA已完全保留在回流液中。
阻断强制再循环,并且通过上述咔唑方法计算收率,该收率等于98%。然后,通过将纯净水引入收集罐,以恒定的体积开始第一个透析步骤,持续时间为使通过UF盒的水量等于9,000升,因此等于罐中存在的体积的两倍。在此步骤中,打开排放阀,并去除9000升透过液,引至特定处理容器。
现在,通过关闭纯净水的供应而排放口仍保持打开状态,将回流液的体积浓缩至初始体积(3,000升)。
用纯净水重复进行透析,直到去除了总计24,000升的透过液,从而完成了8个透析循环。
然后,通过关闭纯净水供应来进行溶液的最终浓缩,至1,000升的体积。然后用纯净水回收产品:保持排放阀关闭,将500升水分批添加到罐中。水在系统内部循环,不断回收回流液并将其重新引入到进料中,以分离出TFF盒中所含的所有HA以及粘附在系统壁上的HA。因此获得1,500升的最终体积,其对于随后的纯化步骤是最佳的,所述纯化根据WO2018/020458的实施例3中所述进行,直到获得干粉形式的最终产物,钠盐形式的透明质酸。
更具体地说,将0.2M NaOH的水溶液添加到回收的产物中,然后将整个混合物用12N HCl中和直到pH达到8.5,最后通过聚丙烯过滤器过滤。HA溶液(现在为钠盐形式)用无水乙醇沉淀,并在搅拌下保持30分钟。将产物静置10分钟,并通过虹吸除去上清液。在连续搅拌下用乙醇:水(80∶20)的混合物洗涤产物30分钟,然后通过虹吸除去上清液。最后的洗涤用无水乙醇进行,该乙醇最终通过过滤除去。将获得的产品放在特殊的不锈钢托盘中,并在25℃的温度下真空干燥至少22小时。
最终产品分析:
蛋白质含量:<0.1%,符合欧洲药典。5.0、1472
细菌内毒素含量:<0.05%,符合欧洲药典。5.0、1472
收率:96.2%
本文提供的数据清楚地表明,将根据本发明目的提取过程的插入到现有技术中已知的通过微生物发酵生产、提取和纯化透明质酸的方法中,令人惊讶地增加了整个方法的收率,从而使得其效率比本领域迄今为止已知的技术大大提高。在这方面,应该记住,被认为尤其有效的已知方法的收率通常在60%至80%之间。
由申请人开发的提取步骤或方法的插入也使整个工艺方法在工业上更为便利。实际上,省去了添加络合盐或吸附树脂,在布或过滤器上的过滤,用有机溶剂反复进行的溶解和沉淀阶段的步骤,从而导致在处理时间和材料使用上以及最终处理废物上节省了净成本。
Claims (16)
1.一种从发酵肉汤中,优选从链球菌(Streptococcus)或芽孢杆菌(Bacillus)属,特别是马链球菌(S.equi)、枯草芽孢杆菌(B.subtilis)或巨大芽孢杆菌(B.megaterium),更优选地马链球菌(S.equi)的微生物的发酵肉汤中提取和纯化HA的方法,其特征在于,它包括提取步骤,该提取步骤包含以下步骤或由以下步骤组成:
a.将过滤的肉汤用纯净水稀释至相对于初始体积的1.1至3体积,优选等于1.5的体积;
b.在包含超滤膜的切向流过滤器(TFF)盒内强制再循环通过合并回流液和透过液形成的来自步骤a.的肉汤,所述超滤膜由芳基磺酸聚合物材料,优选聚醚砜制备,孔隙率范围为5,000至300,000道尔顿,优选50,000至200,000,甚至更优选等于100,000道尔顿,其中重复进行所述强制再循环1-6小时,优选3小时,所述再循环在没有从外部引入液体的情况下以单向流在密闭系统中以恒定体积进行。
2.根据权利要求1的方法,其中在所述强制再循环步骤b.之后进行以下步骤:
c.用选自纯水和盐水溶液的透析溶液,优选纯水进行包含在步骤b.的TFF盒中回流液的透析I;
d.浓缩从步骤c得到的体积至起始肉汤的体积;
e.用选自纯水和盐水溶液的透析溶液,优选纯水进行从步骤d.得到的体积的透析II,重复所述透析5至15次,优选6-12次;
f.最终浓缩从步骤e.得到的体积,直到获得的最终体积等于起始肉汤的体积的三分之一;
g.通过纯水的循环进行保留在TFF盒内的产品的回收,直到获得等于起始肉汤的一半的总体积。
3.根据前述权利要求中一项或多项的方法,其中所述方法的收率为85-100%,优选为95-100%。
4.根据前述权利要求中一项或多项的方法,其中所述纯化的HA的总蛋白质含量<0.1%并且细菌内毒素含量<0.05%。
5.根据前述权利要求中一项或多项的方法,其中所述发酵肉汤是马链球菌马亚种68222,突变体H-1的发酵肉汤。
6.根据前述权利要求中一项或多项的方法,其中所述纯化的HA为碱金属或碱土金属的盐的形式,优选为碱金属,甚至更优选为钠盐的形式。
7.一种从发酵肉汤中,优选从链球菌(Streptococcus)或芽孢杆菌(Bacillus)属,特别是马链球菌(S.equi)、枯草芽孢杆菌(B.subtilis)或巨大芽孢杆菌(B.megaterium),更优选地马链球菌(S.equi)的微生物的发酵肉汤中提取HA的方法,所述方法包含以下步骤或由以下步骤组成:
a.将过滤的肉汤用纯净水稀释至相对于初始体积的1.1至3体积,优选等于1.5的体积;
b.在包含超滤膜的切向流过滤器(TFF)盒内强制再循环通过合并回流液和透过液形成的来自步骤a.的肉汤,所述超滤膜由芳基磺酸聚合物材料,优选聚醚砜制备,孔隙率范围为5,000至300,000道尔顿,优选50,000至200,000,甚至更优选等于100,000道尔顿,其中重复进行所述强制再循环1-6小时,优选3小时,所述再循环在没有从外部引入液体的情况下以单向流在密闭系统中以恒定体积进行。
8.根据权利要求7的方法,其中所述发酵肉汤是马链球菌马亚种,68222,突变体H-1的发酵肉汤。
9.含有根据权利要求1至6中一项或多项纯化的HA的药物、化妆品和营养组合物,用于
治疗关节炎关节、外伤性关节损伤和软骨下损伤;
治疗眼科疾病;
预防术后粘连;
治疗皮肤溃疡、褥疮、烧伤、疤痕和皮肤损伤、瘢痕疙瘩或增生性/肥大性疤痕、各种类型的完整或受损皮肤的皮肤缺陷;
治疗皮肤病,例如湿疹和各种皮炎,特别是特应性皮炎和牛皮癣;
治疗间质性膀胱炎。
10.包含根据权利要求1至6中一项或多项纯化的HA的药物和化妆品组合物,其用于皮肤美学治疗或在美容手术中用作塑形。
11.根据权利要求1至6中一项或多项的化妆品和营养组合物,用于局部和口服。
12.根据权利要求1至6中一项或多项的药物和营养组合物,用于口服治疗关节炎关节,用于肌腱营养,用于皮肤营养和胃肠粘膜营养。
13.根据权利要求1至6中一项或多项纯化的HA,其总蛋白质含量<0.1%并且细菌内毒素含量<0.05%。
14.根据权利要求1至6中一项或多项纯化的HA,为碱金属或碱土金属盐的形式,优选为碱金属,甚至更优选为钠盐的形式。
15.根据权利要求1至6中一项或多项纯化的HA,用于制备HA的衍生物,优选HA与重金属的盐、酯、酰胺、硫酸化和交联产物的衍生物,其中优选自交联产物。
16.根据权利要求1至6中一项或多项纯化的HA,其用于制备为垫、织物、无纺布、颗粒、薄膜和凝胶形式的二维/三维生物材料,还可以与各种来源的细胞和/或血液衍生物,例如血小板衍生物结合。
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IT201800009444A1 (it) | 2018-10-15 | 2020-04-15 | Fidia Farm Spa | Coniugati tra acido ialuronico e amminobisfosfonati e loro impiego terapeutico |
US20220226777A1 (en) * | 2019-05-31 | 2022-07-21 | Asahi Kasei Kabushiki Kaisha | Raw Material Liquid Concentration System |
IT201900019762A1 (it) | 2019-10-24 | 2021-04-24 | Fidia Farm Spa | Composizione farmaceutica per uso nel trattamento della cistite di varia eziologia |
IT202000005692A1 (it) | 2020-03-17 | 2021-09-17 | Fidia Farm Spa | Bio-inchiostro per stampa 3D, relativo coniugato e processo di preparazione di un intermedio costituito da un linker fotoreattivo |
EP3895720A1 (en) * | 2020-04-15 | 2021-10-20 | Euromed, S.A. | Method for obtaining a botanical extract |
IT202000014017A1 (it) | 2020-06-11 | 2021-12-11 | Fidia Farm Spa | Nuovi derivati ottenuti da acido ialuronico e carnosina |
IT202000032243A1 (it) | 2020-12-23 | 2022-06-23 | Fidia Farm Spa | Nuovi agenti antivirali |
IT202100032111A1 (it) | 2021-12-22 | 2023-06-22 | Fidia Farm Spa | Nuovi sostituti biocompatibili dell’umor vitreo |
TR2021022266A2 (tr) * | 2021-12-31 | 2022-01-21 | Irfan Turhan | Fermentasyon yoluyla üreti̇len hyaluroni̇k asi̇t?i̇n fermentasyon ortamindan çevri̇mi̇çi̇ geri̇ kazanimi i̇le üreti̇m veri̇mi̇ni̇n artirilmasi |
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KR102611970B1 (ko) | 2023-12-11 |
WO2019016699A1 (en) | 2019-01-24 |
EA039639B1 (ru) | 2022-02-21 |
PT3655138T (pt) | 2021-07-29 |
AU2018302492B2 (en) | 2023-04-13 |
MA49628B1 (fr) | 2021-09-30 |
CA3062556A1 (en) | 2019-01-24 |
EP3655138B1 (en) | 2021-06-23 |
PL3655138T3 (pl) | 2021-11-22 |
US20190023813A1 (en) | 2019-01-24 |
US11198742B2 (en) | 2021-12-14 |
JP7327748B2 (ja) | 2023-08-16 |
EP3655138A1 (en) | 2020-05-27 |
HUE055703T2 (hu) | 2021-12-28 |
EA201992340A1 (ru) | 2020-03-02 |
ES2883110T3 (es) | 2021-12-07 |
KR20200033223A (ko) | 2020-03-27 |
AU2018302492A1 (en) | 2019-11-07 |
MD3655138T2 (ro) | 2021-10-31 |
MA49628A (fr) | 2021-03-17 |
IT201700081449A1 (it) | 2019-01-18 |
JP2020528000A (ja) | 2020-09-17 |
CN110612153B (zh) | 2022-07-12 |
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