Elsevier

Journal of Chromatography A

Volume 1281, 15 March 2013, Pages 87-93
Journal of Chromatography A

A multimodal histamine ligand for chromatographic purification of plasmid DNA

https://doi.org/10.1016/j.chroma.2013.01.058Get rights and content

Abstract

To exploit different chromatographic modes for efficient plasmid DNA (pDNA) purification a novel monolithic chromatographic support bearing multimodal histamine (HISA) groups was developed and characterized. Electrostatic charge of HISA groups depends on the pH of the mobile phase, being neutral above pH 7 and becoming positively charged below. As a consequence, HISA groups exhibit predominantly ion-exchange character at low pH values, which decreases with titration of the HISA groups resulting in increased hydrophobicity. This feature enabled separation of supercoiled (sc) pDNA from other plasmid isoforms (and other process related impurities) by adjusting salt or pH gradient. The dynamic binding capacity (DBC) for a 5.1 kbp large plasmid at pH 5 was 4.0 mg/ml under low salt binding conditions, remaining relatively high (3.0 mg/ml) even in the presence of 1.0 M NaCl due to the multimodal nature of HISA ligand. Only slightly lower DBC (2.7 mg/ml) was determined under preferentially hydrophobic conditions in 3.0 M (NH4)2SO4, pH 7.4. Open circular and sc pDNA isoforms were baseline separated in descending (NH4)2SO4 gradient. Furthermore, an efficient plasmid DNA separation was possible both on analytical as well as on preparative scale by applying the descending pH gradient at a constant concentration (above 3.0 M) of (NH4)2SO4.

Highlights

► A new chromatographic method for the separation of pDNA isoforms was developed. ► The method employs a multimodal histamine ligand on a monolith. ► Hydrophobic and electrostatic interactions are expressed simultaneously. ► The balance of interaction is determined by the titration state of the histamine. ► Both plasmid isoforms were proven to be selectively eluted on an analytical as well as on preparative scale.

Introduction

The purification strategy of supercoiled plasmid DNA (sc pDNA), a biological macromolecule with increasing pharmaceutical potential [1], [2], [3], involves challenges and constraints that are rarely encountered with other classes of biomolecules [4]. The main reasons are: (1) a low content of pDNA in the cells, (2) the mechanical lability of sc pDNA and (3) impurities sharing common characteristics to pDNA (negative charge – RNA, open circular pDNA (oc pDNA), genomic DNA (gDNA) and endotoxins; high molecular mass – oc pDNA, gDNA and endotoxins; hydrophobicity – endotoxins) [5].

Purification process commonly starts with an alkaline lysis that removes part of the cell walls, organelles, proteins and gDNA, but leaves RNA as the main contaminant together with some proteins, endotoxins and gDNA [6]. Further purification steps can be non-chromatographic e.g., aqueous two-phase extraction [7], tangential flow filtration [8], selective precipitation of pDNA [6], but despite some advantages they are usually followed by a polishing chromatographic step to obtain the product matching the FDA recommendations [9]. Due to these stringent regulatory requests and due to process economics, the majority of downstream processes are based on chromatography. There are different chromatographic separation mechanisms implemented for pDNA purification, such as size-exclusion [10], anion-exchange [11], hydrophobic interaction chromatography (HIC) [12], IMAC [13] or affinity chromatography [14]. However, to completely separate the pDNA from all other impurities on preparative scale, they must be combined in a suitable, robust and efficient purification process. Even the use of highly specific affinity ligands in some cases lacks sufficient selectivity [15] and especially suffers from slow binding kinetics and low recovery of pDNA [16]. Besides, the ligand costs are prohibitively high. A novel approach of immobilizing only 16mer peptide representing helix II of DNA binding domain of lac repressor was recently used in pDNA purification [17], which seems to overcome the above mentioned limitations but on the expense of high ligand costs.

As an alternative, pseudo affinity ligands for pDNA purification are often studied as cheaper substitutes for affinity ligands. Thiophilic chromatography [18] was successfully implemented into a chromatographic process of sc pDNA purification, but it still needed two additional chromatographic steps to complete the plasmid isolation. Certain immobilized antibiotics and anticancer agents binding DNA with high specificity were tested as pseudo affinity ligand as well [19]. An example of such a ligand is immobilized berenil [20], where the purification of pDNA directly from the lysate is achieved in descending (NH4)2SO4 gradient. Its benefits were proven for different plasmid isoforms separation, but no results were shown for the process efficiency on preparative scale. Even certain amino acids, such as lysine, arginine and histidine interact with DNA through multimodal mechanism [21], [22] and they were successfully applied for the separation of pDNA isoforms on an analytical scale.

To achieve high binding capacity, target functionalities have to be introduced onto proper chromatographic support. Monoliths exhibit several advantages over beaded supports when implemented for sc pDNA purification due to large interconnected convective pores and high surface accessibility [23], [24], [25]. The methacrylate based monolithic chromatographic supports have already been successfully implemented for pDNA isoforms separation using a pseudo affinity ligand (imidazole) on a laboratory scale [26]. The main purpose of the present work was to further investigate the performance of amino acid based multimodal ligands. Histamine (HISA), a derivative of amino acid histidine, containing an imidazole ring on a spacer arm was used as a ligand of choice due to its unique chemical properties. Its applicability for the separation of pDNA isoforms under different chromatographic conditions on analytical as well as on preparative scale was studied in detail.

Section snippets

Materials

All solutions were freshly prepared using purified water which meets the requirements for European Pharmacopoeia (AQUATEHNA Biro, Zgornja Kungota, Slovenia) and analytical grade reagents. Buffer solutions were filtered through a 0.22 μm PES filter (TPP, Trasadingen, Switzerland). The samples injected in the column were filtered through cellulose mixed esters syringe filters, pore size 0.45 μm (Macherey-Nagel, Düren, Germany).

Agarose gel was from SeaKem LE Agarose (Lonza Group, Basel,

Histamine modification

The main motive for the research was to find a ligand, presumably with the potential to exhibit both ion-exchange and hydrophobic character that is capable of separating pDNA isoforms on a preparative scale. Different combinations of salt and pH ascending and/or descending gradients were implemented to extensively characterize the selected ligand. Firstly, a multimodal ligand capable of adjusting different ratios between hydrophobic and electrostatic interactions in the vicinity of neutral pH

Conclusions

HISA was immobilized to a monolithic chromatographic support and studied as a multimodal ligand for pDNA purification. In ascending NaCl gradient (preferentially electrostatic interactions) an elution pattern of pDNA was studied in different buffers with the pH value of the buffer as the main changing parameter. Due to the appropriate pKa value of HISA ligand a NaCl concentration for pDNA elution increases considerably from pH 7.4 to pH 5.0. Operating the ligand below pH 6 allows effective

Acknowledgements

We are very grateful to Dr. Fani Sousa and Dr. Angela Sousa from Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, for the fruitful discussions on pDNA pseudo-affinity ligands behavior and for the help in immobilization of amino acids and their derivates to monolithic chromatographic supports. We are greatly thankful to Dr. Gerald Striedner and Dr. Markus Luchner, both from the Microbial Fermentation Group at Department of Biotechnology, BOKU, Vienna, for providing us

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    Presented at the MSS 2012, Ajdovščina, Portorož, Slovenia, 1–6 June 2012.

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