Hanko Valoran P, Rohrer Jeffrey S, Liu Harry H, Zheng Chengmin, Zhang Shaorong, Liu Xiaojie, Tang Xinping
Dionex Corporation, 500 Mercury Drive, Sunnyvale, CA 94088-3603, USA.
J Pharm Biomed Anal. 2008 Aug 5;47(4-5):828-33. doi: 10.1016/j.jpba.2008.04.002. Epub 2008 Apr 8.
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.
用于生产妥布霉素的商业规模发酵是由黑暗链霉菌进行的。在药品生产的各个阶段进行杂质分析对于评估加工步骤的有效性和满足监管要求至关重要。具有集成脉冲安培检测的高效阴离子交换（HPAE）色谱法（HPAE-IPAD）是一种用于测定妥布霉素和评估纯度的高灵敏度方法，但此前没有出版物证明该技术能够在生产过程的各个阶段以典型浓度或典型基质监测纯度。此外，使用HPAE-IPAD分析时，在商业来源的妥布霉素中观察到的杂质峰的身份主要是未知的。监管机构通常要求在发现这些杂质超过一定限度时对其进行表征，而当杂质含量较高时则需要进行毒理学研究。在本文中，我们在生产的不同阶段使用HPAE-IPAD分析妥布霉素样品，并展示了整个制造过程中的杂质谱和浓度变化。基于从我们通过实验重现的极端pH强制降解研究推导的已知降解途径，以及基于在黑暗链霉菌发酵液中发现的先前已知的相关物质，我们成功鉴定了市售妥布霉素中几乎所有的杂质峰。在粗制和最终的妥布霉素产品中，我们鉴定了新霉素、卡那霉素B、暗霉素、卡那胺、2-脱氧链霉胺的峰。我们初步鉴定了粗制和最终产品中的脱氧链霉胺-卡那胺，以及黑暗链霉菌发酵培养下游工艺中间体中的卡那霉素A、氨基甲酰-卡那霉素B和氨基甲酰-妥布霉素。本文给出的结果支持HPAE-IPAD方法在妥布霉素过程中杂质分析中的有效应用，以及作为产品纯化后稳定性指示技术的应用。
