Detection And Control For The Important Impurities Produced During The Fermentation Of Ansamitocin With Strain Actinosynnema Pretiosum Subsp.auranticum

The current fermentation process for ansamitocin,the structural analog of Maytansine is still at a primary stage.Compared with other products which are successfully produced at industry scale fermentation,the quality indicators such as the product purity,relative content of the main product,type and content of impurities are still at a rather low level.In this study,the production of ansamitocin was investigated with Actinosynnema pretiosum subsp.Auranticum ATCC 31565 as the production strain in order to optimize its fermentation process.And mature fermentation process was established on shaking flasks and 10L fermentation tanks,respectively,which was used as a standard for the subsequent production.The initial control level at shaking flask le'vel was:Y3YM medium(maltose 25 g/L;corn dextrin 30 g/L;cottonseed meal 30 g/L;Yeast Extract 5 g/L;calcium chloride 10 g/L;Calcium carbonate 5 g/L;isobutanol 1 g/L;pre-sterilization pH 7.510.2);volume of broth was 30 mL/250 mL;cultivating at 26?/220 rpm for 7 days to obtain the total concentration of ansamitocin at 200?250 mg/L,the percentage of the main product ansamitocin P3(A-P3,one of 5 homologues)was 97%-98%The initial control level at the 10 L fermenter was:After 10 days of fermentation,the total concentration of ansamitocin was 264.88 mg/L,of which P3%was 94%(the fermentation conditions were:Y3YM medium;initial liquid loading,6 L;inoculation amount,8%;initial ventilation,0.5 vvm,gradually increased to 1 vvm according to the level of dissolved oxygen(DO);pressure,0.8?1.2 kg/cm2(adjusted in accordance with the DO level);starting stirring speed,300 rpm,increased with the DO demand,but not above 700 rpm;using KOH to adjust pH 7.2 before inoculation,then,pH not regulated throughout the fermentation process;cultivating at 26? for 10 days).Next,the fermentation broth obtained in the above process was used as a research material,and a standard substance of ansamitocin P3 was used as a reference for comparision.By comprehensive utilization of high performance liquid chromatography(HPLC),liquid chromatography-mass spectrometry(LC-MS),and high Resolution mass spectrometry(HR-MS),nuclear magnetic resonance(NMR)and other analytical methods,the characteristics of ansamitocin P3 and its homologs,as well as the major impurities were located,qualitatively and quantitatively analyzed according to the RT time,molecular weight,and nuclear magnetic resonance spectrum of the characteristic peaks.Among them,it was confirmed that the fermentation broth of this strain contained five ansamitocin homologues including P2/P3/P3'/P4/P4';In addition,8 impurities with relatively large absorption peak area on the spectrum of HPLC were also investigated.The were designated as impurity A-H,and their molecular weights were analyzed one by one.Among them,structures of 5 impurities were fitted by using tools such as ChemDraw.From this,we knew that among the three main impurities of A/B/C,impurity A and impurity B are the intermediates for the deletion of-CH3 and the deletion of-Cl during the biosynthesis of ansamitocin P3,respectively;while the structure of impurity C is temporarily unknown.Because the peak positionof impurity C is very close to the main product P3,which directly affects the purification of P3,it is also listed as one of the most important impurities which should be controlled during the fermentation process.Finally,based on the results of the analysis of the components of the fermentation broth described above,further research focusing on the fermentation control process on the 10 L fermentor and the optimization of its medium composition was carried out in order to control the composition of the ansamitocin homologs(the relative proportion of each component),kinds and quantity of important impurities,and the final goal is to obtain the fermentation broth with high A-P3 proportion and low percentage and content of impurities while ensuring that the total yield of ansamitocin is not reduced.This study finally obtained a main fermentation process for the 10 L scale of fermentation:by using the medium T3,5%inoculum size,continued feeding iso-butanol,300?400 rpm of rotation,0.5?1 vvm of ventilation,incubating at the temperature of 26? for 13 days would result in the total ansamitocin homologs yield as high as 286.29 mh/L,the P3%=95%,and the total relative content of three important impurities of ABC was<5%.With this high-quality fermentation broth,the super-quality assamitocin product could be achieved.The chromatogram purity of the total ansamitocin homologues was 99.9%,of which P3%=99.1%,and the largest single impurity<0.1%,these indicators of quality are much better than those of the standard substance of ansamitocin at current market(The chromatographic purity of all ansamitocin homologs is 90%-95%,and P3%<90%),which has been highly recognized by our international customers.