Datta Payel, Fu Li, Brodfuerer Paul, Dordick Jonathan S, Linhardt Robert J
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Appl Microbiol Biotechnol. 2021 Feb;105(3):1051-1062. doi: 10.1007/s00253-020-11079-9. Epub 2021 Jan 22.
Heparosan is a naturally occurring non-sulfated glycosaminoglycan. Heparosan serves as the substrate for chemoenzymatic synthesis of biopharmaceutically important heparan sulfate and heparin. Heparosan is biologically inert molecule, non-toxic, and non-immunogenic and these qualities of heparosan make it an ideal drug delivery vehicle. The critical-to-quality (CTQ) attributes for heparosan applications include composition of heparosan, absence of any unnatural moieties, and heparosan molecular weight size and unimodal distribution. Probiotic bacteria E. coli Nissle 1917 (EcN) is a natural producer of heparosan. The current work explores production of EcN heparosan and process parameters that may impact the heparosan CTQ attributes. Results show that EcN could be grown to high cell densities (OD 160-180) in a chemically defined media. The fermentation process is successfully scaled from 5-L to 100-L bioreactor. The chemical composition of heparosan from EcN was confirmed using nuclear magnetic resonance. Results demonstrate that heparosan molecular weight distribution may be influenced by fermentation and purification conditions. Size exclusion chromatography analysis shows that the heparosan purified from fermentation broth results in bimodal distribution, and cell-free supernatant results in unimodal distribution (average molecular weight 68,000 Da). The yield of EcN-derived heparosan was 3 g/L of cell free supernatant. We further evaluated the application of Nissle 1917 heparosan for chemical modification to prepare N-sulfo heparosan (NSH), the first intermediate precursor for heparin and heparan sulfate. KEY POINTS: • High cell density fermentation, using a chemically defined fermentation media for the growth of probiotic bacteria EcN (E. coli Nissle 1917, a natural producer of heparosan) is reported. • Process parameters towards the production of monodispersed heparosan using probiotic bacteria EcN (Nissle 1917) has been explored and discussed. • The media composition and the protocol (SOPs and batch records) have been successfully transferred to contract manufacturing facilities and industrial partners.
乙酰肝素是一种天然存在的非硫酸化糖胺聚糖。乙酰肝素是生物制药领域重要的硫酸乙酰肝素和肝素化学酶法合成的底物。乙酰肝素是一种生物惰性分子，无毒且无免疫原性，这些特性使其成为理想的药物递送载体。乙酰肝素应用的关键质量属性（CTQ）包括乙酰肝素的组成、不存在任何非天然部分，以及乙酰肝素的分子量大小和单峰分布。益生菌大肠杆菌Nissle 1917（EcN）是乙酰肝素的天然生产者。当前的工作探索了EcN乙酰肝素的生产以及可能影响乙酰肝素CTQ属性的工艺参数。结果表明，EcN可以在化学限定培养基中生长至高细胞密度（OD 160 - 180）。发酵过程已成功从5升扩大到100升生物反应器。使用核磁共振确认了EcN乙酰肝素的化学成分。结果表明，乙酰肝素的分子量分布可能受发酵和纯化条件的影响。尺寸排阻色谱分析表明，从发酵液中纯化的乙酰肝素产生双峰分布，而无细胞上清液产生单峰分布（平均分子量68,000 Da）。EcN衍生的乙酰肝素产量为每升无细胞上清液3克。我们进一步评估了Nissle 1917乙酰肝素在化学修饰制备N - 磺基乙酰肝素（NSH）中的应用情况，N - 磺基乙酰肝素是肝素和硫酸乙酰肝素的首个中间前体。要点：• 报道了使用化学限定发酵培养基进行益生菌EcN（大肠杆菌Nissle 1917，乙酰肝素的天然生产者）的高细胞密度发酵。• 探索并讨论了使用益生菌EcN（Nissle 1917）生产单分散乙酰肝素的工艺参数。• 培养基组成以及方案（标准操作规程和批次记录）已成功转移至合同生产设施和行业合作伙伴。
