An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1 L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), yield (72% recovery), and productivity (41 mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4mg/ml of membrane volume with a plasmid recovery yield of ∼90%. The effect of the inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance was systematically investigated. Plasmid DNAs with smaller tertiary structure tended to have a better recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other. Membrane pre-treatment significantly improved pore distribution and increased membrane porosity resulting in a better adsorption, recovery, and higher flux. The selection of proper operating pH led to further improvement. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH.

A novel, RNase-free, and potentially scalable bioprocess was developed for the production of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using a high-capacity hydrogel-based strong anion-exchange membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of major impurities of concern for biomedical applications, such as gDNA, proteins, and endotoxin. (C) 2011 Elsevier B.V. All rights reserved.

Trimethylstearylammonium hydroxide/methoxide surfactants were immobilized onto two kinds of cation-exchange membranes (P81 and S2001) for the adsorption of phenolic compounds in the present study. The results indicate that the membrane with a doubled cation-exchange capacity (S2001) could attain nearly twice of the immobilized surfactant amount, but its surfactant immobilization % was close to or lower than the one with a smaller cation-exchange capacity (P81). By manipulating the feed surfactant concentration, different surfactant arrangements on the membrane surfaces (such as hemimicellar, admicellar, or mixed structure) could be produced. The membranes with theoretically 100% surfactant immobilization revealed the highest hydrophobicity level, and thus they were applied in the batch adsorption of phenolic compounds. According to the batch adsorption results of four phenolic compounds onto the surfactant-immobilized membranes (100% immobilization), the main adsorption mechanism should be hydrophobic interaction and the order of phenolic compound adsorptivity was phenol<4-nitrophenol<4-chloro-3-methylphenol≤bisphenol A, identical to their log Kow order. Moreover, in a batch adsorption/desorption cycle with 100mL of 1ppm bisphenol A aqueous sample tested and 5mL of 2-propanol as the desorbent, the S2001 membrane (100% surfactant immobilization) could completely recover bisphenol A from water at a 20-fold enrichment.