Downstream processing of high chain length polysialic acid using membrane adsorbers and clay minerals for application in tissue engineering

Abstract

Polysialic acid (polySia) is a carbohydrate polymer of varying chain length. It is a promising scaffold material for tissue engineering. In this work, high chain length polySia was produced by an E. coli K1 strain in a 10 L bioreactor in batch-and fed-batch mode, respectively. A new downstream process for polySia is presented, based on membrane adsorber technology and use of inorganic anion exchanger. These methods enable the replacement of precipitation steps, such as acetone-, cetavlon-and ethanol-precipitation of the already established purification process. The purification process was simplified, while process efficiency and product qualities were improved. The overall yield of polySia from a 10 L batch cultivation process was 61 % and for 10 L fed-batch cultivation process 40 % with an overall purity of 98 %. The endotoxin content was determined to be negligible (14 EU mg -1 ). Th e ma i n advantage of this new downstream process is that polySia with high chain length > 130 DP (degree of polymerization) can be obtained. In fed-batch cultivation chain lengths up to 160 DP were obtained.

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

“…Deionized water was prepared with Arium ® (Sartorius Stedim Biotech, Göttingen, Germany). For the bioreactor cultivation, a basic defined medium was used as reported previously for a batch cultivation process .…”

“…The concentration of polySia was measured colorimetrically using a modified thiobarbituric acid assay as reported in previous studies . The calibration curve ranged from 0 to 0.75 g L −1 polySia.…”

“…As reported in previous studies, the DP of the produced polySia was analyzed by 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB) HPLC analysis . Therefore, 50 μL sample with a polySia concentration of approximately 1 g L −1 was incubated with 20 μL deionized water, 80 μL TFA (20 mM) and 80 μL DMB solution (7 mM DMB, 12.5 mM Na 2 SO 3 , and 0.8 M β‐mercaptoethanol) for 24 h at 10°C.…”

“…The production of polySia is commonly realized by using bacteria (e.g., E. coli K1 or N. meningitidis B) . The use of bacteria for the biotechnological polySia production has the advantages of being virus free and a fast cell growth with high productivities on cheap media compared to the production with mammalian cell culture.…”

“…This study aims at the development of a novel process using a disposable bag reactor system with wave‐induced mixing for the production of α‐(2,8)‐linked polySia with E. coli K1. The developed process is compared to the state‐of‐the‐art processing in conventional stainless steel reactor setups . The produced polySia was investigated regarding product yield, quality, and chain length.…”