Identification of tobramycin impurities for quality control process monitoring using high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

Commercial-scale fermentation for tobramycin manufacture is carried out with Streptomyces tenebrarius. Impurity profiling during various phases of pharmaceutical production is important for evaluating the effectiveness of a processing step and meeting regulatory requirements. High-performance anion-exchange (HPAE) chromatography with integrated pulsed amperometric detection (HPAE-IPAD) is a highly sensitive method used to assay tobramycin and to assess purity, but no prior publications demonstrated the capability of this technique to monitor purity at various stages of production at either the typical concentrations or in the typical matrices of a manufacturing process. In addition, the identities of the impurity peaks observed in commercial sources of tobramycin when assayed by using HPAE-IPAD are mainly unknown. Regulatory agencies generally require these impurities to be characterized when found above certain limits, and when present at higher levels require toxicological studies. In this paper, we analyze tobramycin samples using HPAE-IPAD at different stages of production and show the impurity profile and concentration changes through the manufacturing process. We successfully identified nearly all the impurity peaks found in commercially available tobramycin, based on known degradation pathways deduced from extreme pH forced degradation studies, which we experimentally reproduced, and based on previously known related substances found in S. tenebrarius fermentation broth. In crude and final tobramycin products, we identified the peaks for neamine, kanamycin B, nebramine, kanosamine, 2-deoxystreptamine. We tentatively identified deoxystreptamine-kanosaminide in crude and final products, and kanamycin A, carbamoyl-kanamycin B and carbamoyl-tobramycin in down stream process intermediates of a S. tenebrarius fermentation culture. Results presented in this paper support the effective use of the HPAE-IPAD method for in-process impurity profiling of tobramycin, and as a stability-indicating technique after product purification.

[Development of new technology for isolation of nebramycin complex antibiotics from the cultural fluid]. / Razrabotka novoi tekhnologii vydeleniia antibiotikov nebramitsinovogo kompleksa iz kurtural'noi zhidkosti.

A new technology for nebramycin complex antibiotics isolation from non-filtered culture fluid was developed. The industrial use of the technology permitted to exclude the stage of the culture fluid filtration, to increase the yield by more than 33% and to reduce the time of the process by 1.7 times.

Saccharocin, a new aminoglycoside antibiotic. Fermentation, isolation, characterization and structural study.

A new aminoglycoside antibiotic, saccharocin has been isolated from the fermentation broth of Saccharopolyspora sp. AC-3440 (FERM P-6238) by column chromatography on a cation-exchange resin. Saccharocin is active against Gram-positive and Gram-negative bacteria. The structure was elucidated to be 4"-deamino-4"-hydroxyapramycin by 13C NMR spectral analysis.

[Isolation and identification of the antibiotics amicetin and nebramycin formed by a Streptomyces coeruleoaurantiacus 4009 culture]. / Vydelenie i identifikatsiia antibiotikov amitsetina i nebramitsina, obrazuemykh kul'turoi Streptomyces coeruleoaurantiacus 4009.

It was found that Str. coeruleoaurantiacus, strain 4009 produced antibiotics 4009-A and 4009-B belonging to different groups. Antibiotic 4009-A was identified as amicetin belonging to the group of pyrimidine bases and antibiotic 4009-B as the nebramycin complex belonging to the group of aminoglycosides. The identity of the antibiotics was confirmed by the physico-chemical constants, spectral and chromatographic characteristics and their chemotherapeutic activity.

Separation of nebramycin components by thin-layer chromatography.

A thin-layer chromatographic method has been developed for the detection of major nebramycin components, for the separation of tobramycin from other components and for studying the hydrolysis of carbamoyl derivatives and procedures for isolation and purification. A sensitive method has also been established for the detection of kanamycin B in tobramycin and for the assay of apramycin in kanamycin B.