Polysialic acid production using Escherichia coli K1 in a disposable bag reactor

Abstract

Polysialic acid (polySia), consisting of α-(2,8)-linked N-acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave-induced mixing.Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor (STR), pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h -1 . Investigation of stress-related product release from the cell surface showed no significant differences between the disposable bag reactor with wave-induced mixing and the STR. After batch cultivation a cell dry weight (CDW) of 6.8 g L -1 and a polySia concentration of www.els-journal.com Page 2 Engineering in Life SciencesThis article is protected by copyright. All rights reserved.

Determination of the Structural Integrity and Stability of Polysialic Acid during Alkaline and Thermal Treatment

“…Manufactured LC polySia [15] with a high endotoxin burden (>3000 EU per mg) was incubated in a sodium hydroxide solution (pH > 13) and recovered via an anion exchange (AEX) membrane adsorber (SartobindQ 75 mL) to fully remove endotoxins. A second parallel experiment used the same initial material for the application of specific endotoxin-removal columns (EndoTrap HD).…”

“…In recent decades, great efforts have been made to improve the production of polySia using E. coli [13,14]. Recent studies have described a complete manufacturing process that also enables the large-scale production of LC polySia (long-chain polySia with a DP ≈ 130) [15,16]. These novel processes are based on disposable systems to facilitate production according to the regulatory requirements of good manufacturing practice (GMP) [17].…”

Development and characterisation of a new fluorescence sensor for online monitoring of bioprocesses

“…The strain used in this study was an Escherichia coli B2032/82 serotype K1. It is an original clinical wildtype isolate (Rode et al, 2008) and is used for polysialic acid production (Vries et al, 2017). The starter culture was prepared by inoculating a glycerol stock into 100 mL lysogeny broth (LB) medium and subsequently incubating at 37 • C, 150 rpm for 7 h. The culture was diluted (1/1000) in 500 mL of defined medium (pH 7.5), which consists of 0.15 g L −1 MgSO 4 q 7H 2 O, 1 mg L −1 FeSO 4 q 7H 2 O, CuSO 4 q 7H 2 O, 9.3 g L −1 K 2 HPO 4 , 2.03 g L −1 KH 2 PO 4 , 10 g L −1 (NH 4 ) 2 SO 4 , 1.2 g L −1 NaCl, 1.1 g L −1 K 2 SO 4 , 13 mg L −1 CaCl 2 and 22 g L −1 glucose.…”

Sensors for disposable bioreactors

“…In many production areas, especially for the production of high‐value products in small volumes, they have replaced traditional stainless steel reactor systems . Disposable reactor technology offers increased facility flexibility with lower investment and energy costs, and especially a simpler production when Good Manufacturing Practice (GMP) requirements are followed, because elaborate and labor‐intensive cleaning procedures become obsolete . Today, various disposable bioreactor systems are available on the market, covering culture volumes from several mL (e.g.…”

“…These systems are stirred, shaken, or pneumatically blended to provide sufficient mixing of all nutrients within the reactor system and to supply an efficient gas exchange into the medium. Although primarily used for mammalian cell culture, the cultivation of yeast and other microorganisms in a disposable bag reactor has been demonstrated [4,6,7]. Overviews of the available systems are given in review articles by Lopez et al [8] and Eibl et al [5,9,10].…”