CN88101111A - 糖肽回收工艺 - Google Patents
糖肽回收工艺 Download PDFInfo
- Publication number
- CN88101111A CN88101111A CN88101111.8A CN88101111A CN88101111A CN 88101111 A CN88101111 A CN 88101111A CN 88101111 A CN88101111 A CN 88101111A CN 88101111 A CN88101111 A CN 88101111A
- Authority
- CN
- China
- Prior art keywords
- antibiotic
- resin
- component
- nutrient solution
- vancomycin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 title description 3
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
一种回收万古霉素型糖肽抗菌素的方法,包括1)使产生抗菌素的发酵培养液与聚苯乙烯二乙烯基苯树脂如Dow XFS-43278.00混合;2)把树脂同培养液分离和3)从树脂上脱抗菌素。本改进方法避免初步过滤和培养液的pH调节,简化了废物处理问题,以及消除了由于菌丝体吸附和过滤而造成的抗菌素损失。
Description
本发明提供一类有价值的抗菌素-万古霉素型糖肽抗菌素回收工艺。万古霉素是50年代后期销售的商业成功的一类抗菌素。例举的万古霉素型糖肽抗菌素包括:
万古霉素(美国专利号3,067,099);M43A(美国专利号4,548,925);M43D(美国专利号4,547,488);M43B和M43C(美国专利号4,548,924);A82846A,A82846B和A82846C(美国申请序列号909,791);瑞斯托菌素(美国专利号2,990,329),瑞斯托菌素A假糖苷配基(Williams等人,J.C.S.Chem.Comm.1979,906-908);A41030因子A-G(美国专利号4,537,770);A47934(美国专利号4,462,942);A35512因子A-D和H(美国专利号4,122,168);A35512B假糖苷配基(美国专利号4,029,769,在该专利中称为A35512B糖苷配基,而由于其还存在氨基糖,所以本文称A35512B假糖苷配基);阿克他菌素(A-4696)因子A和B(美国专利号4,115,552);阿克他菌素因子B1,B2,B3,C1a,C3和E1(美国专利号4,322,406);阿克他菌素因子G(美国专利号4,461,723);阿克他菌素因子H(美国专利号4,558,036);阿克他菌素因子K-O(美国专利号4,479,897);阿克他菌素假糖苷配基(美国专利号4,322,343);磷壁质霉素(teichomycin)A1,A2,和A3(美国专利号4,239,751);磷壁质霉素A因子1-5(美国专利号4,542,018);L17054(美国专利号4,594,187)和L17046(欧洲专利号119,574-A)。
为了便于本文叙述,将这类化合物统称为万古霉素型抗菌素。
万古霉素型抗菌素可用作治疗剂,尤其是作为革兰氏阳性细菌的治疗剂以及也可作为动物生长促进剂。
一般来说,从生产抗菌素的发酵培养液中回收抗菌素时,能采用最少步骤来回收最多的抗菌素。但在大规模生产抗菌素时,大量回收就非常困难。大规模发酵是指在容量至少为约1000加仑(4000升)的容器中发酵。在这种发酵中,必须从大量复杂的发酵混合物的水溶液中分离出抗菌素,由于生产抗菌素的全部发酵培养液不仅含有抗菌素,而且还含有悬浮于未反应营养培养稀释液中的不溶性菌丝体、混杂的代谢中间体和代谢产品。因此,要分离出抗菌素就较困难,需进行大量的分离,浓缩和纯化步骤。
在大多数情况下,回收抗菌素的第一步是借助加入助滤剂例如Hyflo Supercel,来回收经发酵产生的抗菌素。助滤剂的主要好处是分离菌丝体效率高,但进行大规模发酵时,就需要大量的助滤剂。而助滤剂不易进行生物降解,因此大规模使用它们时就存在着这样一个实质问题-废物处理。
除去菌丝体后,常用的第二步是把过滤培养液的pH调到一个适当值来回收经发酵产生的抗菌素,然而这一步需要大量的酸或硷,从而引起操作安全和处理问题,并增加了需处理溶液的体积量。
很显然,在万古霉素的商业制备中,全部培养液是在pH约8至10硷性下进行过滤。然后把滤液的pH调至约6至7,是通过使其通过离子交换树脂。典型的树脂为低交联聚苯乙烯-二乙烯基苯钠阳离子交换树脂,万古霉素被吸附在树脂上。一旦除去培养液后,就用水洗涤树脂,并用pH为9-11的硷性水溶液洗脱万古霉素。典型的洗脱溶剂是pH为10-11的氢氧化钠水溶液。中和含万古霉素的硷性洗脱液,通过把万古霉素再吸附在一种无官能树脂上来纯化该活性组分(参见美国专利4,440,753号)。
本发明的改进方法,包括:1)使产生抗菌素的发酵介质与聚苯乙烯二乙烯基苯树脂如Dow XFS-43278.00相混合,2)分离介质中的树脂,3)洗脱树脂上的抗菌素。本方法的一个优点是发酵培养液无需调节pH和/或过滤就能直接使用。因此,消除了由于抗菌素吸附在菌丝体上和附着在机器上而造成的抗菌素损失。另外,本方法还避免了一些废物的处理问题,例如与助滤剂有关的以及与任何pH调节剂等有关的废物处理问题。
本发明的一个重要方面是采用一种特殊类型的离子交换树脂。这种树脂是一种磺化的苯乙烯和二乙烯基苯的共聚物,为方便起见,这里称作聚苯乙烯二乙烯基苯树脂。该树脂是低交联(约2%)的微孔性强酸性阳离子树脂,其通常是盐的形式如钠盐的形式。适用于这方法的树脂例子为Dow XFS-43278.00(Dow Chemical Co.,Midland,MI,U.S.A.)和Diaion SK-102(Mitsubishi Chemical Industrise,Ltd.,Tokyo,Japan)。我们发现使用这种类型树脂吸收万古霉素型糖肽抗菌素,无需先过滤全部发酵培养液。
可根据方法中发酵培养液的容量和发酵产生的抗菌素活性量的变化,以确定树脂的用量。通常,通过把全部培养液加入树脂或树脂加入全部培养液中,使树脂同全部发酵培养液混合,混合足够时间后,使抗菌素吸附在树脂上。
吸附在树脂上的抗菌素所需接触时间长短是不同的,例如,培养液的温度将影响所需接触时间的长短。在较佳操作中,是把培养液加热到约30℃至60℃,以减少所需接触时间,并降低培养液的粘性,使其易于操作。所需接触时间一般将高达约约6小时。
树脂吸附抗菌素后,采用已知技术,如过滤法(树脂加入分批同培养液接触)或机械分离法(由上流吸附使树脂同培养液混合时)能使吸附有抗菌素的树脂与发酵培养液分离。
接着使用已有操作方法,从分离的树脂中洗脱得抗菌素。更优先的操作方法包括1)水洗树脂,2)通过在pH调至约9至12的水中分批淤浆树脂,直至从树脂上洗脱下抗菌素,3)从树脂上分离出含抗菌素的洗脱液。步骤2)操作中最好是采用10至11的pH值。
从洗脱液中回收抗菌素,并可任意用各种已知方法进一步纯化。例如,可将可溶性抗菌素吸附于无官能树脂(如上所述)上来进一步纯化。另一种方法是作为一种不溶性的铜复合物来分离抗菌素。然后铜复合物在酸性条件下用硫化氢处理,使抗菌素溶解,通过控制合适的pH,以游离硷的形式分离抗菌素。
游离硷形式的抗菌素能用于口服给药的配方中,或将其转化成相应的酸加成盐,例盐酸盐或磷酸盐,这可以用于口服或肠胃外给药。
本发明方法的优点在于可用于任一类的万古霉素型抗菌素,但其用于由发酵方法产生的抗菌素,也就是指万古霉素,43A,B,C和D,A82846A,B和C,瑞斯托菌素,A41030因子A-G,A47934,A35512因子A-D和H,阿克他菌素因子A,B1,B2,B3,C1a,C3,E1,G和H,以及磷壁质霉素A1,A2因子-15和AA等最为适合。本方法尤其适用于万古霉素,因为万古霉素是商业成功并且常常是大规模生产。
下面实施例描述本发明的具体操作。
制备1
抗菌素A82846的制备
(a)产生A82846培养物的发酵
(1)使用NRRL 18098培养物
A.NRRL 18098的摇瓶发酵
用东方奴卡氏菌(Nocardia Orientalis)NRRL 18098培养物,或以其冻干碎片或在液氮中的悬浮液,接种一种含有下列组分的种子培养液:
种子培养液
组分 量%
葡萄糖 1.0
可溶性淀粉 2.0
酵母提取液 0.5
酪蛋白酶水解液 0.5
CaCO30.1
去离子水 1升
无菌前,先用NaOH调节培养液pH值至约7.5。
*Nz按A,Sheffield Chemical Co.,Norwich,NY
2.5%琼脂加入种子培养液制得斜面培养或平板培养。接种的斜面培养在30%孵育约10到14天。成熟的斜面培养物用无菌刀刮松孢子和移动,以及使菌丝体配偶呈绉片(mascerate)。获得的约1/4的松散的孢子和生长培养物被接种到50毫升第一期种子培养液上。
接种的第一期培养液在250ml锥瓶中,于30℃下装在2英寸(5.08厘米)圆周的摇动器轨道上并以250rpm震摇的孵育24-48小时。
孵育的第一期培养液(0.5ml)接种到50ml具有下列组份的生产培养液上:
组分 量%
葡萄糖 2.5
大豆粉 1.5
土豆糊精 3.0
CaCO30.25
废糖密 0.3
酸水解的酪蛋白* 0.5
去离子水 适量1升
(用NaOH调至pH7.5,预先灭菌)
*Hy-Case,Sheffield Chemical Co.
接种的生产培养液在30℃下,于250ml广口锥瓶中孵育4至5天,锥瓶装是在2英寸圆周的摇动器轨道上并于250rpm下震摇的。
B.NRRL 18098的罐发酵
为能提供大量的接种物,由A部分所描述制备的10ml孵育的第一期培养液接种到400ml第二期生长培养液上,这生长培养液含有与第一期培养液相同的组分。这第二级生长培养液在30℃下,于21广口锥瓶中孵育48小时,锥瓶是装在2英寸圆周的摇动器轨道并于250rpm下震摇的。
得到的孵育的第二期生长培养液(1000ml)接种到100升无菌的生产培养液上,该生产培养液按A部分描述的方法制备,只是另加了p-2000消泡剂(S.3g/l)。在30℃下,接种的生产培养液于165升搅拌的发酵罐中发酵90-100小时。调节搅拌器(80RPM)中的空气流使不溶氧气量维持在高于50%的空气饱和度。
C.NRRL 18098的交替式罐发酵
除了使用相应量生长培养液在1600加仑(4536升)发酵罐中接种约1200加仑生产培养液外;按B部分进行操作。
(2)使用NRRL 18099培养物
用东万奴卡氏菌NRRL 18099培养物或以其冻干碎片或在液氮中的悬浮液,按上面描述的(1)部分方法进行培养,只是生产培养液含有下列组分:
组分 量%
葡萄糖 1.0
土豆糊精 2.0
胨* 1.0
CaCO30.2
废糖密 2.0
去离子水不调节ph 适量1升
*Bacto-胨(Difco实验室)
(3)使用NRRL 18100培养物
用东方奴卡氏菌NRRL 18100培养物或以其冻干碎片或在液氮中的悬浮液按上述(1)部分方法进行培养,只是所用的酸水介酪蛋白是Amicase(Sheffield Chemical Co.)。
(b)A82846粗制品的制备
按上述(a)(1)(A)部分的方法,从1600加仑发酵罐制得的发酵培养液(4200升),用5N NaOH调节至pH为10.5,加入3%C盐545(助滤剂)。用压滤机过滤混合物,并水洗压滤机。合并过滤液和水洗液(4200升),用5N HCl(或H2SO4)调节至pH7并通过Dow XFS-43278(NH+ 4)树脂柱(200升滤液/10升树脂)。在750ml/分流速下洗脱柱。用枯草杆菌(Bacillus subtilis)生物测定法或高效液相色谱(HPLC)测定各组分。
用5倍柱体积的水洗涤柱子,收集100升的等分试样。
用5倍柱体积的0.05N NH4OH洗脱柱得活性物质,等分收集25升各组分。合并含A82846的组分并真空浓缩至体积的30升。该溶液加到装有水的10升Diaion HP-20树脂柱中。在300ml/分的流速下用3倍柱体积的水洗涤柱,弃去洗涤水。在流速为100ml/分下,用含有1.0%醋酸的H2O∶i PrOH(95∶5)溶液从柱上洗脱下活性物质,等分收集4升各组分并用生物测定法或高效液相色谱测定。合并含A82846(#6-14)的各组分,真空浓缩和冻干得356克A82846粗制品。
(C)A82846的高效液相测定方法
下面HPLC分析系统用于测定A82846组分:
(1)阳离了交换树脂柱
柱载体:Zorbax*SCX(4.6×150mm)
体系:从A∶B(4∶)至A∶B(1∶9)梯度洗脱5分钟,持续15分钟。
A=MeOH∶0.1M Na H2PO4(1∶9)
B=MeOH∶0.9M NaH2PO4(1∶9)
流速:1∶0ml/分
检测:于225毫微米处紫外光(UV)
保留时间:与浓度有关,但大致约:
A82846C=6.6分
A82846B=8.9分
A82846A=9.5分
(2)反相柱
柱载体:Zorbax*ODS(4.6×150mm)
体系:从1%(NH4)H2PO4∶CH3CN(95∶5)至(1∶1)梯度洗脱20分钟
流速:1.0ml/分
检测:于225毫微米处紫外光(UV)
保留时间:A82846A=7.3分
A82846C=7.6分
A82846B=8.0分
*Zorbax柱是E.I.duPont de Nemours & Co.Inc.,(Wilmington,Delaware 19898)的产品。
(d)A82846组分的分离
(1)A82846A和A82846B的分离
A浓缩的A82846A和A82846B的分离
把按上述(b)部分所制得的A82846(30g)溶于水(500ml)中,并加到有1%NH4H2PO4平衡的硅胶LP-1/C的加压30-L不锈钢柱中。柱子用含1%NH4H2PO4(601)至含1%NH4H2PO4的水∶乙腈(88∶12),在流速为250-300ml/分下梯度展开(最大压为600psi),等分收集4升各组分并使用UV检测器在254毫微米处检测洗出液。各组分用HPLC分析测定。富含A82846A(#6-9)组分和富含A82846B(#10-17)组分各自合并和真空浓缩。
B.A82846A的纯化
把二次各用30克A82846按上面A部分描述所得的富含A82846A浓缩物在装有Diaion HP-20SS的1750ml柱上进行脱盐,然后水洗,用含0.5%乙酸的H2O∶iPrOH(95∶5)洗脱并HPLC分析测定。合并含A82846A各组分,浓缩和冻干得7.4克浓缩的A82846A制剂。
浓缩的A82846A制剂(7.2克)溶于水中,并加到制备性高效液相色谱柱(装有1%(NH4)H2PO4的硅酸LP-1/C18柱)。柱用1%(NH4)H2PO4至1%(NH4)H2PO4∶乙腈(9∶1)进行梯度展开,用HPLC在254毫微米处以48ml/分流速分析检测洗出液。先洗脱10升后,等分收集500ml各组分。
分别合并含A82846A(#4-10)和含A82846B(#12-20)的各组分并真空浓缩。由三次操作得到的A82846A浓缩物合并一起并加到装有Diaion HP-20SS的1750ml柱,对溶液进行脱盐。柱子用水洗后,含0.5%乙酸的H2O∶iPrOH(95∶5)洗脱A82846A。用HPLC测定洗出液,合并含A82846A各组分,浓缩和冻干得7.9克纯化的A82846A。
C.A82846B的纯化
按上述B部分得到并经3次制备性高效液相色谱分离A82846A和A82846B操作的浓缩的A82846B各组分合并一起,在装有Diaion HP-20SS的1750ml柱上进行脱盐,并水洗后用含0.5%乙酸的H2O∶iPrOH(95∶9)洗脱。用HPLC测定洗出液,合并A82846B各组分,真空浓缩和冻干得8.8克纯化的A82846B。
D.脱盐
也可采用Diaion HP-20树脂和含0.1%乙酸的MeOH∶H2O(4∶1)洗脱来完成脱盐。
(2)分离A82846C
A分离A82846
按上述(a)(1)(B)部分方法经4次165升发酵制得的发酵培养液(461升),用5N NaOH调节至pH10.5,并用3%Hyflo Supercel助滤剂过滤。用5N HCl将滤液调至pH7后加到含10升Dowex-XFS-43278(NH+ 4)树脂的柱中,柱用50升水洗,用0.05N NH4OH(50升)洗脱活性物质,等分收集4升各组分并用生物测定法测定洗出液。合并各活性组分(#1-7),真空浓缩至体积约1700ml,然后冻干得283.9克A82846粗制品。
B.A82846A,B和C的分离
按A部分操作得到的A82846粗制品(2g)溶于水中,加到2″×5″的不锈钢制备性HPLC柱中,柱中装有2110ml硅胶LP-1/C18树脂(浸有1%(NH4)H2PO4)。柱用梯度从1%(NH4)H2PO4至1%(NH4)H2PO4∶乙腈(92∶8),流速为70ml/分来展开,等分收集400ml各组分并在254毫微米处紫外光检测。
含A82846A的各组分(#11-14)合并为组合1;含A82846C的各组分(#16-20)合并为组合2,含A82846B各组分(#21-25)合并为组合3。
C.A82846C的纯化
组合2的体积被浓缩至约200ml,并加到装有1800ml Diaion HP-20树脂的7×45cm玻璃柱中进行脱盐。活性物质用含0.1%乙酸的MeOH∶H2O(4∶1)洗脱,以25ml/分流速,收集1升各组分。合并含C(#9-12)的各组分,真空浓缩和冻干得662.2mg半纯化的A82846C。
使用含450ml硅胶LP-1/C的1″×48″不锈钢柱,梯度为1%(NH4)H2PO4至1%(NH4)H2PO4∶乙腈(92∶8),流速为11ml/分的逆相高效液相色谱步骤进一步对半纯化的A82846C(500mg)进行纯化,等分收集25ml各组分并在254毫微米处检测。合并含A82846C组分(#169-210),于HP-20树脂柱(5×45cm玻璃)脱盐。用含0.1%乙酸的MeOH∶H2O(4∶1)洗脱柱,等分收集100ml各组分。接着洗脱液经HPLC的UV在225毫微米分析。合并含A82846C各组分(#5-11),真空浓缩和冻干得127.3毫克A82846C。
按上述同样方法分别纯化含A82846A的组分1和含A82846B的组分3,得到进一步纯化的A82846A和A82846B产物。
D.A82846C的进一步纯化
采用下面制备性色谱操作进一步纯化A82846C(70mg):
柱:Zorbax SCX(9.2×250mm)阳离子交换
树脂流动相:线性梯度,6分钟内从0.15M NaH2PO4缓冲液(含10%MeOH)至0.9M NaH2PO4缓冲液(含10%MeOH),并维持5分钟(不需调节缓冲液)。
流速:6.0ml/分
检测:在280毫微米处的UV
装填:6.0mg/注入水中
装有峰检测装置的自动组分收集器(Gilson201C)用于收集A82846C。流动相由Millipore水M600梯度HPLC系统输送,样品溶液由Hitachi自动样品机注入。
合并含A82846C各组分,浓缩至30ml体积和加入到HP-20柱(50ml)中。柱用H2O洗涤,含0.5%HOAc的H2O∶异丙醇(95∶5)洗脱,分段收集25ml各组分,合并含A82846C各组分(#9-14),浓缩和冻干约37mg纯化的A82846C。
(e)A82846组分的特性
(1)A82846A
分子量:1556
实验式:C73H89N10O26Cl
FAB-MS(硫甘油):(M+1)实测值:1557,5803;
计算值:C73H90N10O26Cl=1557,5716
紫外光谱(H2O)λ最大:281nm(ε5,052),在硷下,位移到300nm。
红外光谱(KBr):1716,1655,1611,1586,1552,1504,1410,
1340,1310,1230,1212,1132,1066,1028和
1015cm-1
PKa(H2O):4.7,9.5
(66%DMF):5.5,6.8,7.9,9.4,12.3
(表观分子量1542)
(2)A82846B
分子量:15%
实验式:C73H88N10O26Cl2
FAB-MS(硫甘油):(M+1)实测值:1591.5315;
计算值:C73H89N10O26Cl2=1591,5327。
紫外光谱(H2O):λ最大:280nm(ε5,192),在硷下位移至300nm。
红外光谱(KBr):1656,1586,1562,1504,1403,1264,1230,
1135,1105,1065,1023和1018cm-1
PKa(H2O):4.65,9.5
(3)A82846C
分子量:1522
实验式:C73H90N10O26
FAB-MS(硫甘油):(M+Na)实测值:1545.59998;
计算值:C73H90N10O26Na=1545.5925
紫外光谱(H2O)λ最大:280nm(ε5,198),在硷下位移至300nm
红外光谱(KBr):3600→3004(宽峰),2999,2991,2950,
1687→1650(宽峰),1585,1570,1509,
1503,1453,1449,1402,1212,1130,1102,
1060,1032和1014cm-1
PKa(H2O):4.6,9.4
(4)其它特性
A82846A,A82846B和A82846C经6N HCl水解后,分析其各自的氨基酸表明,存在天冬氨酸和有痕量甘氨酸存在的二个宽峰。这二个蜂似乎是对应于类放线菌素和万古霉素氨基酸,它们都是存在于万古霉素类的糖肽中的。
NMR相应的研究表明A82846A,A82846B和A82846C各自都含有新的氨基糖4-epi-Vancosamine(3-甲基-acosamine)
A82846A的分子式相对于万古霉素(C66H75N9O24Cl2)来说,少了一个氯原子,但多了一个Vancosamine型(C7H14NO2)的附加氨基糖元素。A82846B的分子式对应于A82846A来说是后者中的氢原子被氯原子取代。A82846C分子式对应于A82846A来说是后者中的氯原子被氢原子取代。因此,A82846组分构成一族新的糖肽类,并且其显然同万古霉素分子的主要组分相同,而主要不同是氯的量,以及在附加亚基(Sub-unit)存在下,具有Vancosamine组分。
(f)A82846抗菌素的抗菌活性
A82846抗菌素在实验室动物的体内显示出对实验引发感染具有抗菌活性。当受试验生物体感染的实验用鼠经双剂量试验化合物给药时,用ED50值〔能起到保护50%试验动物的有效剂量(mg/Kg):可见Warren Wick等,细菌学杂志81卷,233-235页(1961年)〕来测定其活性。化合物所测得的ED50值列于表Ⅱ中。
表Ⅲ:A82846抗菌素的体内活性
金黄色葡萄球菌 ED50值α,生脓链球菌 肺炎链球菌
(Slaphylococcus) (Streptococcus (Streptococcus
化合物 pyogenes) pneumoniae)
A82846A 0.19 0.19 0.17
A82846B 0.19 0.20 0.18
A82846C 2.18 2.71 5.87
万古霉素 1.3 0.72 1.52
αmg/Kg×2;感染1-4小时的老鼠皮下给药剂量
实施例1
万古霉素全部培养液的吸附
把再生的Dow XFS-43278.00树脂(1升)加到含45.4g活性万古霉素的全部培养液中,经室温搅拌6小时后,通过一个100目的筛,使树脂与培养液分离。测定余下的培养液,检查其损耗,弃去余下的培养液。
已吸附的树脂用纯化的水洗涤,然后分批用氢氧化钠洗脱,调节树脂浆液pH至10.5,并维持树脂浆液pH在10.5下搅拌2小时。通过真空过滤从洗脱液中分离出洗脱过的树脂,并用钝化水洗涤。合并收集的洗脱液和水洗液,用不影响溶液稳定性的盐酸调节至pH3.1后进行测定。测定表明回收了37.1g活性体。
洗脱的树脂如下进行再生:用盐酸调节pH至2.0的水溶液中淤浆20分钟,纯化的水洗去过量的酸,在氯作钠溶液中搅拌20分钟,树脂回复到Na+形式,然后用纯化水冲洗除去任何过量盐液。
相反,当使用相同量的全部培养液和采用pH调节和过滤的最佳己有技术回收方案时,在树脂洗脱液中只能得到23.2g万古霉素活性体。
实施例2
M43A全部培养液的吸附
按照美国专利4,548,925,实施例2的方法制造抗菌素M43A,只是B部分不同,省略了下面二步,即步骤1,过滤全部培养液和2)用阳离子交换树脂处理过滤液。而是把再生的Dow XSF-43278.00树脂加到全部培养液中,在室温搅拌该混合物6小时,通过一个筛子把培养液和树脂分离。吸附的树脂按实施例1方法处理回收M43A。
按美国专利4,548,925,实施例2,C部分描述的那样纯化M43A。
实施例3
阿克他菌素全部培养液的吸附
按照美国专利4,322,406的实施例1描述制备阿克他菌素,只是C部分的分离步骤不同,省略了下面步骤,即步骤1)加助滤剂,2)过滤全部培养液,3)把滤块再悬浮于水中,4)调节含水悬浮液pH至10.5,5)过滤。而是把Diaion SK-102加到全部培养液中,使用实施例1中的操作回收阿克他菌素复合物。按本专利实施例1中D和F部分所描述方法分离阿克他菌素因子B1,B2,B3,C1a,C3和E1。
实施例4
磷壁质霉素全部培养液的吸附
按美国专利4,239,751(4-6栏)的描述制备磷壁质霉素,但省略了下列步骤:过滤培养液,调节过滤培养液的pH值,丁醇萃取培养液,在pH3.5下水洗菌丝体块,真空干燥,丙酮水溶液萃取,浓缩丙酮萃取液,调节pH值,以及用丁醇萃取。而是采用本实施例1操作方法回收磷壁质霉素复合物。
实施例5
A82846全部培养液的吸附
按制备1,(a)(3)和(b)部分描述的制备抗菌素A82846,只是采用本实施例1(b)部分的操作来替代下列步骤:调节pH值,加助滤剂,通过压滤机过滤混合物,水洗压滤机和调节合并的过滤液/洗涤液的pH值。
Claims (7)
1、一种回收万古霉素型糖肽抗菌素的方法,其包括:1)使产生抗菌素的发酵培养液与聚苯乙烯二乙烯基苯树脂混合;2)把树脂同培养液分离,3)从树脂上洗脱下抗菌素。
2、根据权利要求1的方法,其中树脂与发酵培养液的混合是通过上流吸收形式把培养液加到树脂中来完成的。
3、根据权利要求1的方法,其中树脂与发酵培养液的混合是通过分批把树脂加到培养液中达到的。
4、根据权利要求1至3的任一方法,其中洗脱溶液为pH9至12的水溶液。
5、根据上面所述权利要求的任一方法,其中抗菌素是万古霉素。
6、根据权利要求1至4中的任一方法,其中抗菌素是选自抗菌素A82846A,A82846B和A82846C。
7、根据权利要求1至4中的任一方法,其中抗菌是选自磷壁质霉素A1,A2和A3,以及磷壁质霉素A2因子1,2,3,4和5。
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US (1) | US4845194A (zh) |
EP (1) | EP0280570B1 (zh) |
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CN (1) | CN1032426C (zh) |
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CN106928323A (zh) * | 2017-03-02 | 2017-07-07 | 重庆乾泰生物医药有限公司 | 一种高纯度奥利万星关键中间体a82846b的制备方法 |
CN106928323B (zh) * | 2017-03-02 | 2021-08-20 | 重庆乾泰生物医药有限公司 | 一种高纯度奥利万星关键中间体a82846b的制备方法 |
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EP0280570B1 (en) | 1996-03-27 |
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EP0280570A3 (en) | 1990-01-24 |
CN1032426C (zh) | 1996-07-31 |
HU199873B (en) | 1990-03-28 |
FI880889A (fi) | 1988-08-28 |
IL85557A (en) | 1993-08-18 |
DE3855145T2 (de) | 1996-09-19 |
ATE136037T1 (de) | 1996-04-15 |
IE880546L (en) | 1988-08-27 |
US4845194A (en) | 1989-07-04 |
FI93228B (fi) | 1994-11-30 |
IL85557A0 (en) | 1988-08-31 |
DE3855145D1 (de) | 1996-05-02 |
EP0280570A2 (en) | 1988-08-31 |
BG47198A3 (en) | 1990-05-15 |
JP2659388B2 (ja) | 1997-09-30 |
JPS63237794A (ja) | 1988-10-04 |
CA1338002C (en) | 1996-01-23 |
FI93228C (fi) | 1995-03-10 |
FI880889A0 (fi) | 1988-02-25 |
GR3019978T3 (en) | 1996-08-31 |
KR970003517B1 (ko) | 1997-03-18 |
KR880009990A (ko) | 1988-10-06 |
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