Andrea Panariello

Application of a 22L scale membrane bioreactor and cross-flow ultrafiltration to obtain purified chondroitin

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed‐batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale‐up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨︁KTA cross‐flow instrument, was translated to a UNIFLUX‐10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C‐3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled‐up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild‐type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity.

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing.

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed‐batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut‐offs for different phases of purification. The operational conditions (cross‐flow rate and transmembrane pressure) used for the process were determined on an ÄKTA cross‐flow instrument (GE Healthcare, USA), a lab‐scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.

Hyaluronic acid degradation during initial steps of downstream processing

Abstract Hyaluronic acid (HA) is a linear glycosaminoglycan composed of disaccharide units of glucuronic acid and N-acetylglucosamine. It has interesting and distinctive viscoelastic properties that are functions of chain length, concentration and environmental conditions, like pH and ionic strength. These characteristics, coupled with its lack of immunogenicity or toxicity, have led to a wide range of applications in the pharmaceutical and cosmetic industries where molecular mass is of primary importance. Biotechnological production of HA was established many years ago, but HA obtained from bacterial fermentation results in shorter chains compared with HA extracted from animal sources. In the present research the issue of HA degradation during the initial phases of downstream processing is addressed. In particular, the effects of clarification and protein separation on molecular weight have been studied using a triple-detector chromatographic system. Environmental factors affecting degradation (pH, shear stresses, etc.) were uncoupled in order to identify the main causes of molecular weight reduction.