Current trends in separation of plasmid DNA vaccines: A review

Chromatographic technology is undoubtedly one of the most diverse and powerful purification methods for downstream process applications. The diversity and quantity of biomolecules present in crude extracts as well as the similarities between impurities and the target biomolecule are considered the critical challenges in the extraction and purification steps. Thus, it is important to optimize the purification protocol to achieve maximum recovery of the target sample. The structure of chromatographic supports has been continuously developed to afford rapid and efficient separations, as well as, the application of specific ligands to improve the selectivity for the target molecule. The present review discusses the structural progress and evolution of the chromatographic supports that have been used for plasmid DNA purification. Nowadays, the most desirable form of plasmid for gene therapy and DNA vaccination is the supercoiled isoform, due to its stability and higher transfection efficiency over other plasmid topologies. However, the main challenge is not only to produce high quantities of supercoiled plasmid DNA but also to preserve its quality, meeting the strict requirements recommended by the regulatory agencies. Therefore, this review will focus on the chemical and structural classification of the different media and on some of the specific ligands used for plasmid DNA bioseparation.

Advances in chromatographic supports for pharmaceutical‐grade plasmid DNA purification

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification.

This chapter first defines the term “whey proteins” as it pertains to the contents of this book. We define whey proteins as the proteins not associated with the casein micelle or other milk particles such as membrane structures and somatic and microbial cells. This chapter divides the whey proteins into two categories of major and minor whey proteins. The major whey proteins discussed here are β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulins. Several characteristics of these proteins are discussed including their abundance in milk of various species, amino acid composition and molecular structure, genetic polymorphism, biological and technological function as well as their significance. The minor whey proteins included in this chapter are lactoferrin, proteose peptone 3, osteopontin, glycomacropeptide, lactoperoxidase, lysozyme, cathepsin D, acid phosphatase, and ribonucleases. In addition to discussing the abundance, characteristics and significance of these minor whey proteins, this chapter summarizes their important features in several useful tables.

Whey Proteins: An Overview

The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices

Biorecognition of supercoiled plasmid DNA isoform in lysine-affinity chromatography.

The recent application of histidine-agarose affinity supports in plasmid purification takes advantage of the biorecognition of nucleic acid bases by the histidine ligand. This consideration prompted the need for better understanding the interactions involved in affinity chromatography of plasmid DNA with the histidine-agarose support. In this work, we used synthetic homo-deoxyoligonucleotides with different sizes (1-30 nucleotides long), to explore the effect of several conditions like hydrophobic character of the individual bases, presence of secondary structures, temperature, pH and salt concentration on the mechanism of retention of nucleic acids to histidine-agarose support. One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention. Otherwise, the temperature manipulation has not shown a direct influence on oligonucleotide retention, only inducing conformational changes on secondary structures. Overall, the results obtained provide valuable information for the future development and implementation of histidine and other amino acids as ligands in chromatography for the purification of plasmid DNA and other nucleic acids, by improving the knowledge of the interactions involved as well as of the parameters influencing the retention.

Histidine affinity chromatography of homo-oligonucleotides. Role of multiple interactions on retention.

A panel of four hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose) was used to examine the selectivity and fractionation of several proteose peptone 3 (PP3) forms from a freeze-dried extract of whey bovine milk. In parti- cular, the effects of altering the ligand type and salt were investigated. The chromatographic studies suggest that PP3 strongly interacts among the three commercial hydrophobic resins leading to a drop off in selectivity, while a complete binding was achieved at low salt concentrations (below 0.5 M) and total elution only with phosphate buffer and/or water stepwise conditions. Only in epoxy-Sepharose was an appreciably selectivity of the several fractions of PP3 present in the initial feedstock attained. Despite the high salt concentration for a complete binding of PP3 (above 1.5 M ammonium sulfate) onto this support, the dual salt system (ammonium sulfate 1 M and sodium citrate 0.8 M) led to a high separation degree of high and low molecular weight forms of PP3. Copyright © 2008 John Wiley & Sons, Ltd.

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A. Sousa

Papers

Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices

Biorecognition of supercoiled plasmid DNA isoform in lysine-affinity chromatography.

Histidine affinity chromatography of homo-oligonucleotides. Role of multiple interactions on retention.

Separation of different forms of proteose peptone 3 by hydrophobic interaction chromatography with a dual salt system

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.