CN102414310B - 用于冷冻干燥乳酸菌的冷冻保护剂 - Google Patents
用于冷冻干燥乳酸菌的冷冻保护剂 Download PDFInfo
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Abstract
本发明涉及一种有效的冷冻保护剂组合物的发现和开发,其不包含脱脂乳或任何其他的动物源性化合物,实现了冷冻干燥的乳酸菌(LAB)在不同温度下的长期稳定性,由此冷冻干燥的乳酸菌(LAB)在贮存6个月、优选贮存9个月及更优选贮存12个月后保留的存活力超过50%。本发明所涉及的领域是生产冻干细菌,特别是乳酸菌。更具体地,本发明涉及冷冻保护剂的新型组合的用途,其用于增加经冷冻干燥后的细菌的存活力、改善冻干的块的质感以易于研磨,以及改善冷冻干燥的细菌在不同温度条件下的长期稳定性。本发明还涉及此类冷冻干燥的细菌,其用于食品工业或用于人类或动物的健康应用。更具体地,本发明涉及重组细菌的提高的存活力和长期贮存能力,所述重组细菌能表达异源蛋白或肽,并以治疗或疫苗接种的目的向人类或动物施用。
Description
本发明涉及冷冻保护剂的新型组合的用途,其用于增加经冷冻干燥后的乳酸菌的存活力、改善冻干的块(cake)的质感以易于研磨,以及改善冷冻干燥细菌在不同温度条件下的长期稳定性。
技术领域
该项发明所涉及的领域是生产冻干细菌,特别是乳酸菌。更具体地,本发明涉及冷冻保护剂的新型组合的用途,其用于增加经冷冻干燥后的细菌的存活力、改善冻干的块的质感以易于研磨,以及改善冷冻干燥的细菌在不同温度条件下的长期稳定性。本发明还涉及此类冷冻干燥的细菌,其用于食品工业或用于人类或动物的健康应用。更具体地,本发明涉及重组细菌的提高的存活力和长期贮存能力,所述重组细菌能表达异源蛋白或肽,并以治疗或疫苗接种的目的向人类或动物施用。
发明背景
乳酸菌(LAB)是一组在分类学上多样的革兰氏阳性细菌,它们能够将可发酵的碳水化合物主要转换成乳酸,从而在该过程中酸化生长培养基。通常,乳酸菌(LAB)种类在食品工业中的用途最为知名,其主要是用于制备发酵食物诸如乳制品和某些肉类。乳制品发酵工业的商业重要性涵盖了例如奶酪、酸乳酪和酸奶油的生产,这些都是世界公认的。
在过去的几十年里,人们对乳酸菌(LAB)的兴趣已显著提高。特定乳酸菌(LAB)菌株能影响肠道生理的这一事实已广泛受到认同。人们对于乳酸菌用作异源蛋白的生产宿主,并最终用作生物活性分子的就地生产和递送系统的兴趣日益增加(见下文)。
目前,研究人员倾注了大量的精力来研究基因改造的(GM)乳酸菌物种用作局部、粘膜给药的生物药物(包括细胞因子、抗体片段、生长因子、激素和神经肽)的生产和递送工具的用途。(例如[6-12])。更具体地,经改造的食品级细菌乳酸乳球菌(Lactococcus lactis(L.lactis))被选为用于治疗性递送生物活性多肽的优选微生物。显然,经改造的乳酸乳球菌菌株用于递送口服治疗性蛋白质的这一概念创造了激动人心的可能性。然而,任何医药产品的一个必要属性是长期稳定性(保质期),在预定的贮存条件下通常为至少24个月。为此,需要为基于经改造的乳酸乳球菌的药物物质(DS)和药物产品(DP)制剂开发一个高效且具伸缩性和可靠性的制造平台。
在制造和后续的贮存期间,产品稳定性的关键参数是经改造的细菌的长期存活力(通常表示为菌落形成单位(CFU)/克,作为贮存时间的函数)。乳酸乳球菌菌株的生产、贮存和最终的治疗使用会对细菌带来显著的压力[4]。在工业环境中,乳酸菌(LAB)可以以液体、喷雾干燥、冷冻或冻干(冷冻干燥)的形式被保存和分配。虽然上述所有制备物都适合用作食品工业中的起始培养物(starter culture),但现在已越来越重视促进高细胞活力和代谢活性的长期保存方法,这是因为这些参数被认为是(生物)药物应用的先决条件。为了最大限度地提高存活能力,向生物质添加所选定的冷冻保护剂以及后续的冻干是关键步骤,特别是考虑到存活的且具有代谢活性的细菌是诱导期望的原位治疗效果的绝对要求这一事实。
冷冻干燥被广泛视为是最适合的细菌脱水过程之一,其目的旨在获得固体的和稳定的最终制剂[4]。其是用于贮存微生物细胞培养物的最常见方法之一,即使冷冻干燥后及贮存过程中的存活率可能因不同菌株而有所不同[5]。冷冻干燥后的存活率反映了细胞抵抗快速冷冻和干燥的影响(例如在数个靶位点处的膜脂质氧化和细胞损伤)的能力[5]。众所周知,大多数无保护的细菌在冷冻干燥后都会被杀死,而存活的那些在进行贮存时迅速死亡。因此,已进行了多种尝试来增加冻干和贮存后存活的细菌的数量,但仅取得有限的成功(见下文)。
冷冻干燥是迄今为止最频繁(如果不是唯一的话)用于实现长期保质期的方法[16]。选择适当的干燥介质/冷冻保护剂混合物是增加乳酸菌(LAB)在冻干和后续贮存过程中的存活率的关键因素[4]。已报告了试图增加乳酸菌(LAB)在冻干和/或后续贮存过程中的存活率的几项研究(关于综述,参见[4])。然而,这些出版物都未证明冻干细菌具有足够的长期稳定性(即一年后能达到>80%的存活率),特别是在室温(25℃)或2-8℃下,如制药应用所需要的。
对于大多数对乳品行业具商业利益的乳酸菌(LAB)培养物而言,脱脂奶粉被选为干燥介质,因为它能够稳定细胞膜的成分、有利于再水化并且形成一层细胞保护膜[4]。补充了额外的冷冻保护剂的脱脂乳可提高其内在的保护作用。
Font de Valdez等描述了在10%脱脂乳中的核糖醇对12株经历冷冻干燥的乳酸菌(LAB)的保护作用[17]。尽管报告显示在冻干过程中获得了高存活率(从42-100%不等,因菌株不同而有所不同),但却没有提供关于长期稳定性的数据。Castro等人评估了脱脂乳(11%)或海藻糖(5%)对保加利亚乳杆菌(Lactobacillus bulgaricus)在冷冻干燥后的存活率的有益效果,相比起在单独的水中的约1%的留存率,它们显示25%的留存率(活细胞计数)[18]。同样地,其没有报导关于后续贮存期间的稳定性的数据。
Carvalho等人(2003)证明了山梨糖醇或谷氨酸(单)钠(MSG)(它们分别被添加至悬浮在脱脂乳中的LAB)对冻干和3-6个月的后续贮存期间的存活的稳定作用[19]。然而,尽管与单独的脱脂乳相比,山梨糖醇或MSG增加了稳定性,但所报导的存活率在山梨糖醇或MSG存在下仍然很低(<0.1%)。此外,在20℃下在空气中贮存于密闭容器中并保存在黑暗之中达8个月的冻干细胞的长期存活率随着时间的流逝而表现出了一个或多个对数的显著下降。
Carcoba和Rodriguez研究了单独添加至重构脱脂乳(RSM)的各种化合物对冷冻干燥后的乳酸乳球菌的细胞存活和代谢活性的影响[16]。他们发现,与单独的RSM相比,糖类的海藻糖和蔗糖、多元醇 的山梨醇和核糖醇、以及氨基酸的β-丙氨酸和谷氨酸都能够把细胞活力增强44.3%以上。然而,未提供补充了培养基的实际存活率,并且没有披露长期贮存数据。
作为最后一个示例,Huang等人所进行的研究开发和优化了德氏乳杆菌(Lactobacillus delbrueckii)的保护介质,其在冷冻干燥后导致86%的细胞存活力[20]。该介质的组成是:66.40克/升的蔗糖、101.20克/升的甘油、113.00克/升的山梨醇以及130.00克/升的脱脂乳。同样地,该研究并没有报道有关长期稳定性的结果。
Huyghebaert等人旨在开发一种含有具可接受保质期的存活的GM乳酸乳球菌的冻干粉剂制剂[21]。为了调查冷冻干燥基质的影响,他们使用了两种不同的介质:补充有0.5%葡萄糖的M17肉汤(以获得GM17),或补充有0.5%葡萄糖和0.5%酪蛋白水解物的10%(w/v)脱脂乳(以获得GC-乳)。在冷冻干燥之后,他们评估了冻干参数、冷冻干燥基质和不同贮存条件对短期和长期存活力的影响。
当在常规GM17肉汤中进行冻干时,绝对存活力低于10%,而在GC-乳基质内进行的冻干则导致显著较高的存活力(60.0±18.0%)。然而,尽管多次尝试标准化冷冻干燥过程,但仍无法避免批次间的显著的变异性。
短期稳定性研究的结果显示,存活力在冷冻干燥和1周的贮存后(GC-乳基质)已下降±20%。在长期稳定性研究中,相对存活力在1个月的贮存后高度下降,并且在随后几个月的贮存期间对数递减(GC-乳基质,各种贮存条件),这表明无法获得长期稳定性。
整体地考虑现有技术,很明显的是,脱脂乳是乳酸菌(LAB)冷冻干燥介质的反复出现的组成部分,因此它对细菌的存活力似乎是必不可少的。然而,乳衍生物在新型药物组合物的使用是严格被禁止的,特别是考虑到与其使用相关的传染性海绵状脑病(TSE)风险。
除了生产后的高存活力,冻干的乳酸菌(LAB)在制药应用领域方面还应具有可接受的长期保质期。例如,US 2005/0100559、US 3897307和WO2004/065584描述了稳定的干燥的细菌组合物。US 2005/0100559 中的干燥细菌组合物的特点是它们含有很大比例的稳定剂。参见例如US 2005/0100559中的实施例[055],其中稳定剂占了至少40%(w/v)。在WO2004/065584中,蔗糖或蔗糖和麦芽糖糊精显示能提高细菌细胞培养物的稳定性,但前提是必须在-20℃下。在该文献中没有教导如何改善含有冻干细菌的组合物的长期保质期(在室温下)。在US3897307中,所有实验都从在脱脂乳中的不同乳酸菌物种的培养物开始,其随后进行冻干,任选地在稳定增强剂存在下,所述稳定增强剂选自L-抗坏血酸(包括其可食用的盐),和谷氨酸或天门冬氨酸(包括其盐)。因此,乳成分是稳定的干燥细菌组合物的一个重要组成部分。
换句话说,现有技术均未指出,更换乳成分能在长期贮存下获得足够的存活和稳定性。事实上,大多数此类研究都缺乏关于初始存活力、稳定性和细菌密度的精确数据。最后,这些研究都没有报告GM细菌的冷冻干燥和/或其性质的维持。
附图简述
图1:乳酸乳球菌菌株sAGX0037在使用不同冷冻保护剂混合物的情况下的活细胞计数(以菌落形成单位[CFU]/g表示):紧在冷冻干燥后及在暴露于25℃/35%RH 24小时后的数据。表1描述了冷冻保护剂混合物的详细组成。
图2:乳酸乳球菌菌株sAGX0037在使用不同冷冻保护剂混合物的情况下的活细胞计数(以菌落形成单位[CFU]/g表示):紧在冷冻干燥后及在暴露于25℃/35%RH 24小时后的数据。表3描述了冷冻保护剂混合物的详细组成。
图3:乳酸乳球菌菌株sAGX0037在使用冷冻保护剂混合物Z4(20%谷氨酸钠,10%山梨醇和10%葡聚糖500)进行冷冻干燥并且最终贮存温度为25℃和35℃的情况下的活细胞计数(以菌落形成单位[CFU]/g表示):紧在冷冻干燥后及在暴露于25℃/35%RH 4和24小时后的数据。表4描述了细菌和冷冻保护剂混合物组合物的详细组成。
图4:谷氨酸钠、糊精(来自玉米淀粉)和山梨醇在冷冻干燥和长 期贮存(在3种不同的贮存条件下)期间对乳酸乳球菌菌株sAGX0037的稳定作用:-20℃;5℃及25℃/60%RH,在PET/ALU袋中。表5描述了细菌和冷冻保护剂混合物组合物的详细组成。
发明详述
本发明涉及一种有效的冷冻保护剂组合物的发现和开发,其不包含脱脂乳或任何其他的动物源性化合物,实现了冷冻干燥的乳酸菌(LAB)在不同温度下的长期稳定性,由此冷冻干燥的乳酸菌(LAB)在贮存6个月、优选贮存9个月及更优选贮存12个月后保留的存活力超过50%、优选超过60%,更优选超过70%,更优选超过80%。冷冻保护剂组合物制剂的主要优点是在冷冻干燥期间、在短期暴露于正常生产操作条件期间及在包装后的长期贮存期间,为高度敏感的细菌提供保护。
本发明提供了稳定剂(冷冻保护剂)的组合,其在冷冻干燥、研磨和筛分冻干的块以及后续的贮存时获得乳酸菌(LAB)的高存活。为了最大限度地提高存活率,在冷冻干燥前将不同的稳定化合物(冷冻保护剂)的组合添加到细菌生物质。该稳定化合物组合包括淀粉水解物和谷氨酸盐和/或多元醇,导致冻干乳酸菌(LAB)的改善的存活率和稳定性。具体地,本发明涉及冷冻保护剂的新型组合的用途,其用于增加经冷冻干燥后的细菌的存活力、改善冻干的块的质感以易于研磨,以及改善冷冻干燥细菌在不同温度条件下的长期稳定性。
如下文实施例详细解释的,在所选的冷冻保护剂混合物的存在下在冷冻干燥后获得高存活细胞产率(>6x 10E+11菌落形成单位[CFU]/g)。令人惊讶的是,这些冻干细胞的存活力并没有因接触环境条件(模拟下游药物配制和生产过程[例如:胶囊灌装])长达24小时(25℃/35%RH)而受到影响,并且在不同的贮存条件下也观察到了细胞存活力的长期保存。
冷冻保护剂组合包含谷氨酸钠或山梨醇和葡聚糖,同时结合了诸如海藻糖和蔗糖等的公知冷冻保护剂,其在冷冻干燥后得到与优选的 冷冻保护剂制剂(代码D)相当的活细胞产率。短期暴露研究清楚地显示,包含淀粉水解物和谷氨酸盐和/或多元醇的此类组合的冷冻保护剂制剂在未受保护的贮存下在25℃和35%RH下保护冻干乳酸乳球菌长达24小时。在本发明中,谷氨酸盐优选是谷氨酸钠。本发明的多元醇优选是山梨醇或甘露醇,而本发明的淀粉水解物则优选是葡聚糖。
乳酸乳球菌(L.lactis)菌株sAGX0037的存活分析的结果通过冻干样品及暴露于空气中的样品的存活细胞计数来确定,其表明,淀粉水解物(如葡聚糖500)、谷氨酸钠和多元醇(如甘露醇)的优选组合(代码D)(如实施例1、2和3所示),在未受保护的贮存下在25℃和35%RH下保护冻干乳酸乳球菌长达24小时。
相较于蔗糖制剂(其也称为用于稳定冻干乳酸菌(LAB)的“金标准”),上述3种冷冻保护剂的组合在贮存方面显然有更优越的表现,而且也是唯一能在短期暴露于25℃/35%RH下导致>6x10E+11CFU/g的活细胞计数的稳定剂组合。当谷氨酸钠被单独添加至细菌时,在短期暴露后,没有观察到细菌的存活。
淀粉水解物(如来自玉米淀粉的糊精)、谷氨酸钠和多元醇(如山梨醇)的冷冻保护剂组合能产生稳定的冻干乳酸菌(LAB)粉末,从而保证了活细菌的长期稳定性和存活率(在研磨和筛分贮存于-20℃及5℃的冻干的块时,没有观察到任何显著的活细胞计数下降,并且在1年的贮存后也有>90%的最初活细胞计数得以保留,导致非常高的CFU浓度,高达>5x 10E+11CFU/g)。在25℃及60%RH下,只观察到轻微的活细胞计数下降,仍然得到非常高的CFU浓度,在25℃及60%RH下贮存1年后仍高达>3x 10E+11CFU/g。
因此,本发明的目的是提供冷冻干燥的细菌组合物,其包含淀粉水解物、谷氨酸盐和多元醇的组合。从下文的实施例中可知,所述组合物中所用的淀粉水解物的量为约2.0%到约10%(w/v);特别是约2.5%至约5%(w/v)。所述组合物中所用的谷氨酸盐的量为约2.0%到约10%(w/v);特别是约5.0%至约7.5%(w/v)。所述组合物中所用的多元醇的量为约5.0%到约30%(w/v);特别是约10%至约20%(w/v),更具体来说为约7.5%至约15%(w/v)。
本文中所使用的“多元醇”通常是指数种糖醇的混合物,如山梨醇、麦芽糖醇、甘露醇等等。它是由玉米淀粉、马铃薯淀粉或小麦淀粉(其被淀粉酶分解成小单位,如葡萄糖、糊精、麦芽糊精和聚糊精(polydextrin)等等)水解而成。在随后的氢化步骤里,所述的小单位会被转换成糖醇,如山梨醇、麦芽糖醇、甘露醇和较长链的氢化糖类(如maltitriitol)。在本发明的具体实施方案中,多元醇是甘露醇、山梨醇或山梨醇和甘露醇的组合。在所述实施方案中,每个所述多元醇,即山梨醇或甘露醇,各自独立地以约5.0%至约15%(w/v),特别是约7.0%至约15%(w/v)的量存在。在进一步的实施方案中,每个所述多元醇成分以相同的量存在,即约7%、8%、9%、10%、11%、12%、13%、14%或15%(w/v)。
本文中所使用的“谷氨酸盐”通常是指谷氨酸和其可食用的水溶性盐。这类“可食用的”盐是被批准用于人类食品中且是食品级的那些,如谷氨酸的钠和/或钾盐。在本发明的优选实施方案中,谷氨酸盐是谷氨酸单钠,也被称为谷氨酸钠及MSG。在进一步的实施方案中,所述谷氨酸钠以约2.0%到约10%(w/v);特别是约5.0%至约7.5%(w/v)的量存在。
本文中所使用的“淀粉水解物”通常是指包含大量葡萄糖单位的支链多糖如淀粉或葡聚糖等的水解产物。淀粉的构建单位包括线性和螺旋的直链淀粉及分枝的支链淀粉。葡聚糖的直链包含葡萄糖分子之间的α-1,6糖苷键,而支链则开始自α-1,4键(在某些情况下,α-1,2及α-1,3键)。在具体实施方案中,本文中所使用的淀粉水解物包含下列任何一种:葡聚糖1、葡聚糖5、葡聚糖10、葡聚糖20、葡聚糖40、葡聚糖60、葡聚糖70、葡聚糖110或葡聚糖500,其中数字是指以kDa表示的规范性分子量;在更具体的实施方案中,淀粉水解物为葡聚糖500。在进一步的实施方案中,所述葡聚糖以约2.0%至约10%(w/v),特别是约2.5%至约5%(w/v)的量存在。
如在本发明的说明书和实施例中使用的,单数形式“一种”、“一个”和“该”包括单数和复数所指物,除非上下文另外明确说明。举例来说,“细胞”是指一个或超过一个的细胞。
本文中所使用的术语“含有”、“包含”和“包括”是同义词,其是包容性或开放式的,不排除额外的、未被引述的成员、元素或方法步骤。
用端点表示的数值范围包括该范围内所包含的所有数值及分数,以及所引述的端点。
当描述一个可测量的值,例如参数,量,时间期限等时,本文中所使用的术语“约”意欲涵盖与指定值相差+/-20%或更少、优选+/-10%或更少、更优选+/-5%或更少、更优选+/-1%或更少,更优选+/-0.1%或更少的变化,此类变化适宜在所披露的发明中使用。
本说明书中列举的所有文件以其全文通过引用并入本文。具体来说,本文中特别提到的所有文件的教导通过引用并入本文。
除非另有说明,用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导,随后的定义用于更好地理解本发明的教导。
术语“重组核酸”通常是指,包含利用重组DNA技术连接在一起的区段的核酸。当重组核酸在宿主生物体内复制时,其后代核酸也包含在术语“重组核酸”内。
陈述重组DNA技术的一般原则的标准参考著作包括Molecular Cloning:A Laboratory Manual,2nd ed.,vol.1-3,ed.Sambrook et al.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989;Current Protocols in Molecular Biology,ed.Ausubel et al.,Greene Publishing and Wiley-Interscience,New York,1992(定期更新)(″Ausubel et al.1992″);Innis et al.,PCR Protocols:A Guide to Methods and Applications,Academic Press:San Diego,1990。微生物学的一般原则陈述于例如Davis,B.D.et al.,Microbiology,3rd edition,Harper & Row,publishers, Philadelphia,Pa.(1980)中。
当提及给定的ORF与启动子之间的关系时,术语“异源的”表示,所述启动子通常不与所述ORF天然结合,也即,通常不天然控制所述ORF的转录。换句话说,这些结合是通过重组DNA技术在本发明的重组核酸中形成的。
“乳酸菌(lactic acid bacterium)”这一术语通常是指,选自乳球菌属(Lactococcus)物种、乳杆菌属(Lactobacillus)物种、链球菌属(Streptococcus)物种、片球菌属(Pediococcus)物种、双歧杆菌属(Bifidobacterium)物种和明串珠菌属(Leuconostoc)物种的细菌,并且包括本领域内归类为上述的任何类群(如物种、亚种、株系)。
“乳球菌”这一术语通常是指乳球菌属细菌,并且包括本领域内归类为该属的任何类群(如物种、亚种、株系)。作为例子,乳球菌包括格氏乳球菌(Laxtococcus garvieae)、乳酸乳球菌(Laxtococcus lactis)、鱼乳球菌(Lactococcus piscium)、植物乳球菌(Lactococcus plantarum)和棉子糖乳球菌(Lactococcus raffinolactis),以及其亚种和株系。
在本发明的优选实施方案中,乳球菌是乳酸乳球菌。乳酸乳球菌包括但不限于,乳酸乳球菌乳脂亚种(Lactococcus lactis ssp.cremoris)、乳酸乳球菌霍氏亚种(Lactococcus lactis ssp.hordniae)、乳酸乳球菌乳亚种(Lactococcus lactis ssp.lactis)及乳酸乳球菌酸双乙酰乳变种(Lactococcus lactis ssp.bv.diacetylactis)。
在本发明的进一步优选的实施方案中,乳酸乳球菌是乳酸乳球菌乳脂亚种或乳酸乳球菌乳亚种,更优选为乳酸乳球菌乳亚种,并且包括其任何株系,如乳酸乳球菌乳脂亚种SK11或乳酸乳球菌乳亚种MG1363。
因此,在一个实施方案中,冻干细菌包含一个或多个编码表达产物(优选多肽)的重组核酸开放阅读框,所述表达产物能够在受试者优选人或动物受试者中引起治疗应答。
在特别有用的示例性且非限制性的实施方案中,本发明所述的一个或多个重组核酸开放阅读框可编码抗原和/或非疫苗原性的(non-vaccinogenic)治疗活性多肽。
本文中所使用术语“抗原”通常是指能激发免疫应答(包括体液免疫和/或细胞免疫应答)且能够结合免疫应答的产物(如抗体或T细胞)的对于生物体(尤其是对于被给予抗原的人类或动物受试者而言)是外源的物质。因此,在一个优选的例子中,抗原需要被给予所述抗原的受试者具有功能性免疫系统,以引起生理应答。
根据本发明的抗原可来源于人或动物受试者对其的免疫应答在治疗上有用的任何多肽,如来自病原体,如来自病毒、原核生物(如细菌)或真核病原体,来自非生理性蛋白(如源自癌组织的蛋白质),来自变应原(如用来激发免疫耐受性)等。
术语“非疫苗原性的治疗活性多肽”通常是指在施用其的人或动物受试者中不引起抗其自身的免疫应答且能实现治疗作用的多肽。因此,预期此类多肽的治疗效果与其自身的天然生物学功能直接相关,由此其可以在受试者体内达到特定的效果,而不是作为受试者体内的免疫原性和/或免疫保护性抗原而产生治疗效果。因此,非疫苗原性的治疗活性多肽在其表达形式上应具有生物学活性,或至少在从表达宿主细胞中释放时转换成生物学活性形式。所述多肽的生物学活性形式优选能展示二级构型,或更优选地三级构型,所述构型与其天然构型相同或非常类似。
非疫苗原性的治疗活性多肽优选是无毒和非病原性的。
在优选实施方案中,非疫苗原性的治疗活性多肽可来源于人类或动物,且优选对应于与待施用其的人类或动物受试者相同的类群。
合适的非疫苗原性的治疗活性多肽的非限制性例子包括,能够局部或全身性起作用的多肽,例如能够施加内分泌活性来影响局部或全身代谢的多肽,和/或生物学活性多肽是能够调节免疫造血系统的细胞活性的多肽,和/或一个或多个生物学活性多肽是能够影响体内各种正常或肿瘤细胞的存活力、生长和分化或影响对损伤和感染的急性期炎 性应答的免疫调节或诱导的多肽,和/或一个或多个生物学活性多肽是能够增强或诱导对细胞和组织的感染的抗性(通过作用于其靶细胞受体的趋化因子介导)或上皮细胞的增殖或促进伤口愈合的多肽,和/或一个或多个生物学活性多肽能够调节体内细胞的物质表达或生产。
此类多肽的具体例子包括但不限于,胰岛素,生长激素,促乳素,降钙素,促黄体激素,甲状旁腺激素,生长抑素,促甲状腺激素,血管活性肠肽,细胞因子如IL-2,IL-3,IL-4,IL-5,IL-6,IL-7,IL-9,IL-10,IL-11,IL-12,IL-13,IL-14至IL-32的任一个,GM-CSF,M-CSF,SCF,IFNs,EPO,G-CSF,LIF,OSM,CNTF,GH,PRL,细胞因子的TNF家族,如TNFa,TNFb,CD40,CD27或FAS配体,细胞因子的IL-1家族,成纤维细胞生长因子家族,血小板源性生长因子,转化生长因子和神经生长因子,细胞因子的表皮生长因子家族及胰岛素相关细胞因子等。另外,治疗活性多肽可以是上述定义的治疗活性多肽的受体或拮抗剂。此类合适多肽的进一步具体例子列举于例如,以引用方式并入本文中的WO 96/11277第14页第1-30行;以引用方式并入本文中的WO 97/14806第12页第1行到第13页第27行;或以引用方式并入本文中的US 5,559,007第8栏第31行到第9栏第9行。
因此,在一个实施方案中,重组核酸编码抗原和/或非疫苗原性的治疗活性多肽,其中所述的抗原能够引发人体或动物受试者的免疫应答,优选保护性免疫应答,和/或所述的非疫苗原性的治疗活性多肽能够在人或动物受试者体内产生治疗作用。
WO 97/14806进一步具体地披露了抗原与免疫应答刺激分子(例如白细胞介素,诸如IL-2或IL-6)的细菌共表达。因此,还预期本发明的冻干细菌用于此类共表达。
在进一步优选的实施方案中,根据本发明的开放阅读框进一步包含编码分泌信号的序列,该序列与ORF所编码的多肽同相。这有利地允许表达的多肽从宿主细胞分泌,从而可促进例如分离或递送。
在通常情况下,分泌信号序列代表约16到约35个氨基酸的区段,其通常包含嵌入到脂质双层膜中的疏水氨基酸,从而允许所伴随的蛋 白质或肽序列从宿主细胞分泌,并且其通常从该蛋白质或肽切除。优选,分泌信号序列在意欲用于包含所述信号序列的核酸的宿主细胞(例如细菌宿主细胞,优选乳酸菌,更优选乳球菌,而更优选乳酸乳球菌)内是有活性的。
在合适的宿主细胞中有活性的分泌信号序列是本领域已知的;示例性乳球菌信号序列包括,usp45的信号序列(请参阅US 5,559,007)和其他信号序列,请参阅,例如,Perez-Martinez et al.1992(Mol Gen Genet 234:401-11);Sibakov et al.1991(Appl Environ Microbiol 57(2):341-8)。优选,信号序列位于启动子序列及ORF之间,即,信号序列位于启动子序列的3′端和目的多肽的ORF之前。在优选的实施方式中,信号序列编码如下氨基酸序列:
MKKKIISAILMSTVILSAAAPLSGVYA(usp45)。
另一方面,本发明的冻干细菌含有包含重组核酸的载体。
本文所使用的“载体”是指一种核酸分子,其通常为DNA,并且其中可插入和克隆(即增殖)核酸片段。因此,载体通常包含一个或多个独特的限制性酶切位点,并能够在确定的宿主或媒介生物体内自主复制,从而克隆的序列是可复制的。载体可包括但不限于质粒、噬菌粒、噬菌体、噬菌体衍生载体、PAC、BAC、线性核酸、如线性DNA等等(请参阅例如Sambrook et al.,1989;Ausubel 1992)。
本发明的重组核酸或载体可以以染色体外的形式存在于宿主细胞内,优选其使用自身的复制起点进行自主复制,或其可整合入细菌基因组DNA,如细菌染色体,例如乳球菌染色体。对于后一种情况,可整合所述核酸的一个或多个拷贝,优选单个拷贝;整合可发生在染色体的随机位点,或如上文所述,发生在预定的位点,优选在预定的位点,例如在优选的非限制性例子中,在乳球菌例如乳酸乳球菌的thyA基因座。
在相关方面,本明提供了一种生产冻干细菌的方法,所述冻干细菌内的重组核酸含有一个或多个开放阅读框,用于给有此需要的人或动物施用,包括给所述人或动物施用治疗有效量的用所述核酸转化的 所述细菌。
动物优选是哺乳动物,如驯养动物、农场动物、动物园动物、体育动物、宠物和实验动物,如狗、猫、豚鼠、兔子、大鼠、小鼠、马、牛、母牛;灵长类动物如猿、猴子、猩猩和黑猩猩;犬科动物如狗和狼;猫科动物如猫、狮和虎;马科动物如马,驴和斑马;食用动物如牛、猪及羊;蹄类动物如鹿和长颈鹿;啮齿类动物如小鼠、大鼠、仓鼠和豚鼠等。
本文中所使用的术语“治疗”或“疗法”是指治疗性治疗和预防性或防止性措施,其中目标是为了防止或减慢(减少)不想要的生理变化或障碍。“需要治疗的人类或动物”包括将受益于给定治疗条件的那些。
术语“治疗有效量”是指,有效治疗诸如人类或动物等受试者的疾病或障碍的治疗性物质或组合物的量,即获得期望的局部或全身效果和性能的量。例如,治疗有效量的细菌可能包含至少1个细菌、或至少10个细菌、或至少有102个细菌、至少103个细菌、或至少有104个细菌、或至少105个细菌、或在至少106个细菌、或至少有107个细菌、或至少有108个细菌、或至少109、或至少1010、或至少1011、或至少1012、或至少1013、或至少1014、或至少1015或更多的宿主细胞,如细菌,例如在单个或重复的剂量中。
本发明的冻干细胞可单独施用,或者与一个或多个活性化合物组合施用。后者可以在施用所述冻干细胞之前、之后或同时施用。
许多现有技术公开了抗原和/或治疗活性多肽的递送,因此应理解,这些公开内容可进一步有利地使用本发明的强启动子进行改善。例如但不限于,三叶肽的细菌递送可用于治疗消化道疾病(请参阅例如WO 01/02570)、白细胞介素特别是IL-10的递送可用于治疗结肠炎(请参阅例如WO 00/23471)、抗原的递送可作为疫苗(请参阅例如WO 97/14806)、GLP-2和相关类似物的递送可用于治疗短肠疾病、克罗恩病、骨质疏松症及作为癌症化疗期间的辅助治疗,等等。可设想使用本发明的冻干细胞的其他治疗应用。
可通过本发明的治疗性多肽的递送治疗的人类或动物疾病类型的其他非限制性例子包括但并不限于,包括克罗恩病和溃疡性结肠炎在内的炎性肠道疾病(可用例如IL-lra或IL-10或三叶肽治疗);自身免疫疾病,包括但不限于银屑病,类风湿性关节炎,红斑狼疮(可用IL-lra或IL-10治疗);神经病症,包括但不限于阿尔茨海默病,帕金森病和肌萎缩性侧索硬化(可用例如脑源性神经营养因子和睫状神经营养因子治疗);癌症(可用例如IL-1、集落刺激因子或干扰素-W治疗);骨质疏松症(可用例如转化生长因子f3治疗);糖尿病(可用例如胰岛素治疗);心血管疾病(可用例如组织型纤溶酶原激活剂治疗);动脉粥样硬化(可用例如细胞因子和细胞因子拮抗剂治疗);血友病(可用例如凝血因子治疗);退化性肝脏疾病(可用例如肝细胞生长因子或干扰素a治疗);例如囊肿性纤维化的肺部疾病(可用例如α抗胰蛋白酶治疗);肥胖;病原体感染,如病毒或细菌感染(可用任何上述组合物或抗原治疗);等等。
另一方面,本发明因此还提供了包含本发明所制造的冻干细菌的药物组合物,无论该冻干细菌是否用上文所述的核酸和/或载体进行了转化。
所述制剂优选包含治疗有效量的本发明所制造的冻干细菌及药学上可接受的载体,即一个或多个药学上可接受的载体物质和/或添加剂,例如缓冲剂、载体、赋形剂、稳定剂等等。本文中所使用的术语“药学上可接受的”在本领域内的含义是一致的,意思是指与药物组合物的其他成分相容,且对受者无害。本发明的冻干细菌可以制成胶囊剂、片剂、颗粒剂和粉剂形式,而所有的这些形式都可通过口服途径给药。
此外,本发明的冻干细菌也可制成在合适介质内的水性悬浮液,或者可紧在使用前将冻干细菌悬浮在合适的介质中。
对于口服施用,可配制肠溶制剂的口服剂型,该剂型还可包括提供宿主细胞的控制释放的化合物,从而能提供其中编码的所需蛋白质的控制释放。例如,口服剂型(包括片剂、弹丸剂、颗粒剂、粉剂)可 以涂上赋形剂薄层(通常是聚合物、纤维素衍生物和/或亲脂性材料),其能抵抗在胃内而非在肠道内的溶解或破坏,从而允许通过胃部到达肠道进行崩解、溶解和吸收。
口服剂型可被设计成允许宿主细胞或其重组蛋白质的缓慢释放,例如控制释放、缓释、延长释放、长效片剂或胶囊。这些剂型通常包含常规和广为人知的赋形剂,如亲脂性赋形剂、聚合物赋形剂、纤维素赋形剂、不溶性赋形剂、膨胀赋形剂。控释制剂也可用于任何其他递送位点,包括肠、结肠、生物粘附或舌下递送(即口腔粘膜递送)和支气管递送。当本发明的组合物将通过直肠或阴道进行给药时,药物制剂可包括栓剂和药膏剂。在此实例中,将宿主细胞悬浮在常见赋形剂的混合物(其还包括脂质)中。上述所提到的各种制剂在本领域中是众所周知的,并且描述在例如下列文献中:Hansel et al.(1990,Pharmaceutical dosage forms and drug delivery systems,5th edition,William and Wilkins);Chien 1992,Novel drug delivery system,2nd edition,M.Dekker);Prescott et al.(1989,Novel drug delivery,J.Wiley & Sons);Cazzaniga et al.,(1994,Oral delayed release system for colonic specific delivery,Int.J.Pharm.i08:7′)。
灌肠剂制剂优选可用于直肠给药。“灌肠剂”这一术语用来涵盖意欲用于直肠的液体制剂。灌肠剂通常可以提供于单剂量容器中,并包含一种或多种溶解或分散于水、甘油或聚乙二醇(macrogol)或其他合适溶剂中的活性物质。
因此,根据本发明,在优选实施方式中,编码所需基因的重组宿主细胞可通过粘膜例如口、鼻、直肠、阴道或支气管途径,通过可用于所述途径的任何现有技术制剂,向动物或人类施用。施用的宿主细胞的剂量会因各种因素而有所不同,所述因素包括细菌及其所编码的基因的类型、待治疗的疾病的类型和严重程度以及所使用的给药途径。
因此,对于本发明的每一个和所有实施方案,无法界定精确的剂量,但对于本领域技术人员来说,在阅读本发明后,其将是显而易见 的。总之,剂量将按照个案方式而具体确定:使用广为人知的方法(如ELISA或Biacore,请参阅实施例)来测量在施用预定数目的细胞后重组蛋白的血清浓度。所递送的重组蛋白的动力学特点及半衰期的分析将提供足够的信息来确定经转化的宿主细胞的有效剂量范围。
在一个实施方案中,当按照本发明制造的冻干细菌表达抗原时,本发明也可提供疫苗。
“疫苗”这一术语是指药学上可接受的组合物,其当以有效量施用给动物或人类受试者时,能在受试者中诱导针对疫苗内包含的免疫原的抗体和/或激发保护性免疫。
本发明的疫苗包含用本发明的核酸或载体转化的宿主细胞,及任选地赋形剂。此类疫苗还可包括佐剂,即增强对抗原的免疫应答的化合物或组合物。佐剂包括但不限于,完全弗氏佐剂、不完全弗氏佐剂、皂甙、矿物凝胶例如氢氧化铝、表面活性物质例如溶血卵磷脂、嵌段多元醇、聚阴离子、肽、油或烃类乳剂和潜在有用的药学上可接受的人佐剂,如BCG(卡介苗)和短小棒状杆菌(Corynebacterium parvum)。
本发明的冻干乳酸菌可用于一般食品工业,用作食品添加剂或特别是用作起始培养物,例如酸乳酪起始培养物或奶酪起始培养物。通常情况下,这些组合物包含浓缩形式的细菌,包括冷冻的、干燥的或冷冻干燥的浓缩物,其活细胞浓度通常为至少10<5>CFU/克组合物,如至少10<6>CFU/克,包括至少10<7>CFU/克,如至少10<8>CFU/克,如至少10<10>CFU/克,如至少10<11>CFU/克,如至少10<12>。组合物可包含常规添加剂(包括营养物质,例如酵母提取物、糖和维生素)作为进一步的成分。
本发明提供了一种制造冻干的乳酸菌的方法,所述乳酸菌可用于各种食用产品的组分或成分,例如乳包括非巴氏杀菌的乳(生乳)、肉、面团、酒和植物材料,例如蔬菜、水果或饲料作物等。本文中所使用的术语“乳”是指任何类型的乳或乳成分,例如牛乳、人乳、水牛乳、山羊乳、绵羊乳、由此类乳制成的乳制品、或乳清。本发明的冻干乳酸菌的特别的优势是,具有高存活力和长期贮存能力。起始培养物以 这样的量添加,所述量导致下列活细胞数:至少10<3>菌落形成单位(CFU)/克食用产品起始材料,如至少10<4>CFU/克,包括至少10<5>CFU/克,如至少10<6>CFU/克,如至少10<7>CFU/克,如至少10<8>CFU/克,如至少10<9>CFU/克,如至少10<10>CFU/克,如至少10<11>CFU/克,如至少10<12>/克食用产品起始材料。
本发明还提供了一种包含本发明的稳定化合物的组合的冻干乳酸菌,其为用于生产食品或动物饲料的起始培养物组合物的形式,或用于产生香气的培养物的形式。
本发明通过不被认为是限制性的实施例作进一步的说明。
实施例
通过参考下列实验细节,将更好地理解本发明,但本领域技术人员易于理解,这些都只是本发明的示例,本发明由之后的权利要求更充分地描述。基于这些实施例,其他实施方案对于本领域技术人员来说是显然的,特别是包括其他乳酸菌的实施方案,所述乳酸菌例如乳球菌属物种,例如乳杆菌属物种、链球菌属物种、片球菌属物种、双歧杆菌属物种、明串珠菌属物种以及本领域内归类为上述的任何类群(如物种、亚种、株系)。
实施例1:葡聚糖、谷氨酸钠和多元醇的组合在冷冻干燥期间和短期(24小时)暴露于25℃/35%RH期间所发挥的稳定作用
将乳酸乳球菌菌株sAGX0037培养在5升的发酵罐内,并用含有800μM胸苷的经纯化的水清洗2次,然后浓缩十倍。浓缩的细菌以1∶1的体积比与冷冻保护剂混合物(1毫升样品+1毫升冷冻保护剂)混合。所产生的制剂以2毫升的体积被分装进35个小瓶内。从生物反应器到小瓶的整个过程花费约8个小时,且平均温度为10℃。具有最终浓度的各种制剂(即包括细菌在内)如表1所示。将小瓶冷冻于固体二氧化碳颗粒中,直到将它们置于冷冻干燥机中为止。
表1:加入细胞悬浮液后的冷冻保护剂制剂的组成。配制的细胞悬浮液中细胞的干重含量为33克/升或3.3%。
为了测试冻干的块在暴露于环境条件(模仿下游配制过程[如胶囊充填])后的存活率,分析3个小瓶的紧在冷冻干燥后以及在25℃和35%RH下贮存24小时后的活细胞计数。
乳酸乳球菌菌株sAGX0037的存活分析的结果通过冻干样品及暴露于空气的样品的活细胞计数来确定,结果表明淀粉水解物(如葡聚糖500)、谷氨酸钠和多元醇(如甘露醇)的组合(如代码D所示),在未受保护的贮存下在25℃和35%RH下保护冻干的乳酸乳球菌达24小时。
虽然与公知的冷冻保护剂例如海藻糖和蔗糖组合的包含谷氨酸钠或山梨醇和葡聚糖的组合(制剂代码A,B和C),在冷冻干燥后得到与代码D制剂相当的活细胞产率(>1x 10E+11CFU/g),但短期暴露研究清楚地显示,仅包含淀粉水解物和谷氨酸盐和/或多元醇的组合的制 剂D在未受保护的贮存下、在25℃和35%RH下、在24小时期间保护冻干的乳酸乳球菌。
相较于蔗糖制剂(代码E)(即,用于稳定冻干乳酸菌(LAB)的“金标准”),所选的组合(D)在贮存方面有更优越的表现,而且也是本实施例中唯一在短期暴露于25℃/35%RH后产生>1x 10E+11CFU/g的活细胞计数的组合。
如果细菌没有添加稳定剂,那么如图1,代码F制剂所示的,在短期暴露后没有观察到细菌的存活。
实施例2:葡聚糖、谷氨酸钠和多元醇的组合在冷冻干燥期间和短期(24小时)暴露于25℃/35%RH期间所发挥的稳定作用
预先培养的乳酸乳球菌菌株sAGX0037(100毫升)用于接种7LContinuously Stirred Tank Reactor(CSTR),其含有5升的M17c培养基(组成列于表2中)。
表2:M17c发酵培养基的组成
组分 | 数量(用于1升) |
酵母提取物 | 20克 |
KH2PO4 | 2.0克 |
MgSO4,7H2O | 0.51克 |
柠檬酸一水合物 | 0.49克 |
葡萄糖 | 55克 |
胸苷(100mM) | 8毫升/升 |
生物反应器被设置成将温度保持在30℃及将pH保持在7(通过添加5M的NH4OH)。搅拌速度设为200rpm。当葡萄糖消耗完成时,通过将发酵罐冷却至4℃来终止发酵。取出“发酵结束时”的样品,用于测定细胞干重(DCW)。在终止发酵后,浓缩3.5升的发酵肉汤,然后通过使用1400cm2 500kDa的中空纤维过滤器的超滤/渗滤来进行清洗。
在总量3.5升的发酵肉汤被浓缩约10倍后,将5升体积的瓶替换 成含经纯化的水的瓶,用于渗滤。在渗滤过程中,通过分析渗透物来监测乳酸的浓度。当乳酸浓度达到5-10克/升时,终止渗滤。
紧在浓缩和渗滤后,将细菌细胞悬浮液分为13份,然后分别加入不同的冷冻保护剂(列于表3)。在混合细胞悬浮液及冷冻保护剂后,将每种混合物分装进超过25个小瓶(2毫升终体积)并冻干。
表3:加入细胞悬浮液后的冷冻保护剂制剂的组成。配制的细胞悬浮液中细胞的干重含量为70克/升。
为了测试冻干的块在暴露于环境条件(模仿下游配制过程[如胶囊充填])后的细菌存活率,分析3个小瓶的紧在冷冻干燥后以及在25℃和35%RH下贮存24小时后的活细胞计数。
乳酸乳球菌菌株sAGX0037的存活分析的结果通过冻干样品及暴露于空气的样品的活细胞计数来确定,结果表明淀粉水解物(葡聚糖500)、多元醇(如甘露醇和/或山梨醇)和谷氨酸钠的组合(如代码D1所示),紧在冷冻干燥后得到高活细胞计数(如>3E+11CFU/g),并且在未受保护的贮存下在25℃和35%RH下稳定冻干的乳酸乳球菌达24小时,得到高活细胞产率(>2E+11CFU/g)。
虽然代码为A1、A2、B1、C1和C2的制剂(与公知的冷冻保护剂例如海藻糖和蔗糖组合的包含谷氨酸钠或山梨醇和葡聚糖的组合)紧在冷冻干燥后得到与代码D1制剂相当的活细胞产率,但短期暴露研究清楚地显示,制剂D1和D2(包含至少淀粉水解物(葡聚糖500)、多元醇(如甘露醇和/或山梨醇)和谷氨酸钠的混合物)在未受保护的贮存下、在25℃和35%RH下、在24小时期间稳定冻干的乳酸乳球菌。
相较于蔗糖制剂(代码E1)(即,用于稳定冻干乳酸菌(LAB)的“金标准”),上述3种冷冻保护剂的组合在贮存方面有更优越的表现,而且也是本实施例中唯一在短期暴露于25℃/35%RH后产生>2x10E+11CFU/g的活细胞计数的稳定剂组合。
当谷氨酸钠被单独添加到细菌中时,如图2,代码F2制剂所示的,在短期暴露后没有观察到细菌的存活。
实施例3:葡聚糖、谷氨酸钠和山梨醇的组合在冷冻干燥期间和 未受保护的短期(4和24小时)暴露于25℃/35%RH期间所发挥的稳定作用
预先培养的乳酸乳球菌菌株sAGX0037(100毫升)用于接种7LCSTR,其含有5升的M17c培养基(见上文表2)。生物反应器被设置成将温度保持在30℃及将pH保持在7(通过添加5M的NH4OH)。搅拌速度设为200rpm。当葡萄糖浓度降至低于0.5克/升时,通过将发酵罐冷却至4℃来终止发酵。取出“发酵结束时”的样品,用于测定DCW。在终止发酵后,浓缩3.5升的发酵肉汤,然后通过使用1400cm2 500kDa的中空纤维过滤器的超滤/渗滤来进行清洗。
在总量3.5升的发酵肉汤被浓缩约10倍后,将5升体积的瓶替换成含经纯化的水的瓶,用于渗滤。在渗滤过程中,通过分析渗透物来监测乳酸的浓度。当乳酸浓度达到5-10克/升时,终止渗滤。
将细菌悬浮液与不同的冷冻保护剂混合在一起,其组成可参见表4。在混合悬浮液及冷冻保护剂后,将每种混合物在无菌条件下分装进不同的冷冻干燥容器(55毫升等分)中。在分装后,将容器放置在平板上,然后放入-70℃的冰箱直到冻干为止。在冷冻干燥过程期间评估两个次级的干燥温度:25℃及35℃的贮存温度。
表4:加入细胞悬浮液后的冷冻保护剂制剂的组成(70克/升的干重)。
乳酸乳球菌菌株sAGX0037的存活分析的结果(图3)通过冻干样品及暴露于空气的样品的活细胞计数来确定,结果表明淀粉水解物(葡聚糖500)、多元醇(如甘露醇和/或山梨醇)和谷氨酸钠的组合,紧在冷冻干燥后得到高活细胞计数(如>6E+11CFU/g),并且在未受保护的 暴露于25℃和35%RH下稳定冻干的乳酸乳球菌达4小时和24小时。
在本次暴露测试中观察到了高活细胞稳定性,4小时暴露后及24小时暴露后的活细胞产率分别为>6E+11CFU/g及>5E+11CFU/g。
表4a:冷冻保护剂制剂在冻干后对乳酸乳球菌存活率的影响
实施例4:糊精、谷氨酸钠和山梨醇的组合在冷冻干燥期间和不同贮存条件下的长期贮存期间所发挥的稳定作用
在200升规模的乳酸乳球菌菌株sAGX0037的工业发酵后,将累积的生物质浓缩,并分别用纯化的水通过超滤/渗滤进行清洗。当乳酸浓度低于5克/升(55毫摩尔/升)时,终止渗滤。在超滤和渗滤期间,将容器护套持续以水冷却至4℃。
在随后的冻干步骤中,通过向获自UF/DF步骤的生物质加入选定的冷冻保护剂溶液来确保细菌的稳定性。最终的冷冻保护剂溶液包含淀粉水解物(来自玉米淀粉的糊精)、谷氨酸钠和多元醇(山梨醇),如表5所述。
表5:冷冻保护剂制剂的组成
组分 | 重量 |
糊精(来自玉米淀粉) | 100克=10%w/v |
山梨醇 | 100克=10%w/v |
谷氨酸钠 | 200克=20%w/v |
用于注射的水 | QSP 1L |
所添加的冷冻保护剂溶液的所需重量为约17.0千克的细胞悬浮液和4.8千克的冷冻保护剂溶液,因此最终制剂的重量为约21.8千克 配制的细胞悬浮液。该制剂被分配到合适的冻干托盘内,然后在经验证和监控的条件下进行冷冻干燥。托盘被置入冷冻干燥机内的架子上,随后被冷冻至-50℃。总冷冻时间约为9小时。
在冷冻步骤后,降低舱室的压力,并通过增加贮存温度(以直至-22℃、-10℃、20℃、25℃及35℃(最终贮存稳定)的多重坡道步骤)来开始初级干燥。在初级干燥结束时,进行压力上升测试以确定初级干燥期结束。没有发生压力上升的情况,因此通过二级干燥期继续冷冻干燥过程。冷冻干燥过程的总计时间为约93小时。
冷冻干燥周期结束时,用干燥、无菌过滤的氮气(在具有0.22微米孔径的膜上过滤)来给舱室加压。拿出托盘并将其分别装入18-26℃及30%-70%RH下的防蒸汽铝(Alu)箔袋内。
然后,将冻干的块放入温度(19-23℃)及湿度(RH<20%)受控制的100.000级生产室内平衡一小时,然后通过手工研磨(在PE袋中)冻干的块并进行筛分(410微米)来使其粉碎。在筛分后,手工均质化粉末(在PE袋中),然后从最终的冻干的药物物质(DS,其含有乳酸乳球菌sAGX0037和冷冻保护剂)取出样品,用于进行分析和稳定性测试。
将样品以每500毫克装入PET/Alu袋,并贮存于-20±5℃、5±3℃和25±2℃,60±5%RH下。监测样品12个月。如图4所示,在贮存于-20℃及5℃的粉化的冻干细菌中没有观察到显著的活细胞计数下降,在1年的贮存期间保持>90%的初始活细胞计数,从而得到非常高的CFU浓度(高达>5x 10E+11CFU/g)。在25℃及60%RH下只观察到轻微的活细胞计数下降,仍然得到非常高的CFU浓度,在25℃及60%RH下贮存1年后CFU浓度仍高达>3x 10E+11CFU/g。
这些数据清楚地表明,淀粉水解物(如来自玉米淀粉的糊精)、谷氨酸钠和多元醇(如山梨醇)的冷冻保护剂组合产生稳定的冻干乳酸菌(LAB)粉末,确保了活细菌的长期稳定性和存活率。
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Claims (18)
1.用于改善冷冻干燥的乳酸菌的存活率和稳定性的稳定化合物的组合,其包含淀粉水解物和谷氨酸盐和多元醇,其中所述多元醇是甘露醇或山梨醇,所述淀粉水解物是葡聚糖或者糊精;并且,其中:
-所述淀粉水解物的量为2.0%到10%(w/v);
-所述谷氨酸盐的量为2.0%到10%(w/v);并且
-所述多元醇的量为5.0%到30%(w/v)。
2.根据权利要求1所述的稳定化合物的组合,其中所述谷氨酸盐是谷氨酸钠。
3.根据权利要求1或2所述的稳定化合物的组合,其中所述淀粉水解物是由葡萄糖单位组成的支链多糖的水解产物。
4.根据权利要求1所述的稳定化合物的组合,其中糊精包括麦芽糊精和/或聚糊精。
5.根据权利要求1所述的稳定化合物的组合,其包含来自玉米淀粉的糊精。
6.根据权利要求1所述的稳定化合物的组合,其中所述淀粉水解物是葡聚糖。
7.根据权利要求1所述的稳定化合物的组合,其包含淀粉水解物和谷氨酸钠和多元醇,其中所述多元醇是甘露醇或山梨醇,所述淀粉水解物是葡聚糖或者糊精;并且,其中:
-所述淀粉水解物的量为2.0%到10%(w/v);
-所述谷氨酸钠的量为2.0%到10%(w/v);并且
-所述多元醇的量为5.0%到30%(w/v)。
8.根据权利要求7所述的稳定化合物的组合,其中糊精包括麦芽糊精和/或聚糊精。
9.根据权利要求7所述的稳定化合物的组合,其包含来自玉米淀粉的糊精。
10.冷冻干燥乳酸菌的方法,其中使用根据权利要求1至9任一项的稳定化合物的组合。
11.一种组合物,其包含冷冻干燥的乳酸菌,以及权利要求1至9任一项的稳定化合物的组合。
12.根据权利要求11的组合物,其中所述乳酸菌还包含一种或多种重组核酸,所述重组核酸对所述细菌是异源的。
13.根据权利要求11或12的组合物,其用作药物或食品成分。
14.根据权利要求11或12的组合物,其用于食品应用或食品加工。
15.根据权利要求14的组合物,其为用于生产食品或动物饲料的起始培养物组合物的形式,或为用于生产香气的培养物的形式。
16.根据权利要求1或2所述的稳定化合物的组合,其中所述乳酸菌选自乳球菌属(Lactococcus)物种、乳杆菌属(Lactobacillus)物种、链球菌属(Streptococcus)物种、片球菌属(Pediococcus)物种、双歧杆菌属(Bifidobacterium)物种和明串珠菌属(Leuconostoc)物种。
17.根据权利要求10的冷冻干燥乳酸菌的方法,其中所述乳酸菌选自乳球菌属(Lactococcus)物种、乳杆菌属(Lactobacillus)物种、链球菌属(Streptococcus)物种、片球菌属(Pediococcus)物种、双歧杆菌属(Bifidobacterium)物种和明串珠菌属(Leuconostoc)物种。
18.根据权利要求11或12的组合物,其中所述乳酸菌选自乳球菌属(Lactococcus)物种、乳杆菌属(Lactobacillus)物种、链球菌属(Streptococcus)物种、片球菌属(Pediococcus)物种、双歧杆菌属(Bifidobacterium)物种和明串珠菌属(Leuconostoc)物种。
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