Versatility of polymethacrylate monoliths for chromatographic purification of biomolecules

Polymethacrylate monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate) or poly(GMA-co-EDMA) monoliths, are a new generation of chromatographic supports and are significantly different from conventional particle-based adsorbents, membranes, and other monolithic supports for biomolecule purification. Similar to other monoliths, polymethacrylate monoliths possess large pores which allow convective flow of mobile phase and result in high flow rates at reduced pressure drop, unlike particulate supports. The simplicity of the adsorbent synthesis, pH resistance, and the ease and flexibility of tailoring their pore size to that of the target biomolecule are the key properties which differentiate polymethacrylate monoliths from other monoliths. Polymethacrylate monoliths are endowed with reactive epoxy groups for easy functionalization (with anion-exchange, hydrophobic, and affinity ligands) and high ligand retention. In this review, the structure and performance of polymethacrylate monoliths for chromatographic purification of biomolecules are evaluated and compared to those of other supports. The development and use of polymethacrylate monoliths for research applications have grown rapidly in recent times and have enabled the achievement of high through-put biomolecule purification on semi-preparative and preparative scales.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Using DNA as a drug - bioprocessing and delivery strategies

DNA may take a leading role in a future generation of blockbuster therapeutics. DNA has inherent advantages over other biomolecules such as protein, RNA and virus-like particles including safety, production simplicity and higher stability at ambient temperatures. Vaccination is the principal measure for preventing influenza and reducing the impact of pandemics; however, vaccines take up to 8-9 months to produce, and the global production capacity is woefully low. With production times as short as 2 weeks, improved safety and stability, bioprocess engineering developments, and the ability to perform numerous therapeutic roles, DNA has the potential to meet the demands of emerging and existing diseases. DNA is experiencing sharp growths in demand as indicated by its use in gene therapy trials and DNA vaccine related patents. Of particular interest for therapeutic use is plasmid DNA (pDNA), a form of non-genomic DNA that makes use of cellular machinery to express proteins or antigens. The production stages of fermentation and downstream purification are considered in this article. Forward looking approaches to purifying and delivering DNA are reported, including affinity chromatography and nasal inhalation. The place that pDNA may take in the preparation for and protection against pandemics is considered. If DNA therapeutics and vaccines prove to be effective, the ultimate scale of production will be huge which shall require associated bioprocess engineering research and development for purification of this large, unique biomolecule.

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Peptides constructed from α-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to "biomimics" of the DNA binding domain of LacI. Equilibrium dissociation constants (K D), association (k a), and dissociation rates (k d) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10 -10 M, 7.2 ± 0.6 × 10 -10 M, 4.5 ± 0.5 × 10 -8 M, and 6.2 ± 0.9 × 10 -6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon-repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Rapid production of a plasmid DNA encoding a malaria vaccine candidate via amino-functionalized poly(GMA-co-EDMA) monolith

Malaria is a global health problem; an effective vaccine is urgently needed. Due to the relative poverty and lack of infrastructure in malaria endemic areas, DNA-based vaccines that are stable at ambient temperatures and easy to formulate have great potential. While attention has been focused mainly on antigen selection, vector design and efficacy assessment, the development of a rapid and commercially viable process to manufacture DNA is generally overlooked. We report here a continuous purification technique employing an optimized stationary adsorbent to allow high-vaccine recovery, low-processing time, and, hence, high-productivity. A 40.0 mL monolithic stationary phase was synthesized and functionalized with amino groups from 2-Chloro-N,N- diethylethylamine hydrochloride for anion-exchange isolation of a plasmid DNA (pDNA) that encodes a malaria vaccine candidate, VR1020-PyMSP4/5. Physical characterization of the monolithic polymer showed a macroporous material with a modal pore diameter of 750 nm. The final vaccine product isolated after 3 min elution was homogeneous supercoiled plasmid with gDNA, RNA and protein levels in keeping with clinical regulatory standards. Toxicological studies of the pVR1020-PyMSP4/5 showed a minimum endotoxin level of 0.28 EU/m.g pDNA. This cost-effective technique is cGMP compatible and highly scalable for the production of DNA-based vaccines in commercial quantities, when such vaccines prove to be effective against malaria. © 2008 American Institute of Chemical Engineers.

Binding mechanism of affinity ligands for purification of plasmid DNA

Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity chromatography makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. In this study, surface plasmon resonance (SPR) based Biacore system has been employed for the detection and quantification of the binding between lac operon (lacO) sequence contained in a pDNA and synthetic peptides based on the DNA-binding domain of the lac repressor protein, lad. The equilibrium dissociation constant (K D) and association and dissociation rate constants (ka, kd) for the interaction between plasmid DNA and designed peptides were determined.

Fingerprint analysis of eucommia bark by LC-DAD and LC-MS with the aid of chemometrics

Chromatographic fingerprints of 46 Eucommia Bark samples were obtained by liquid chromatography-diode array detector (LC-DAD). These samples were collected from eight provinces in China, with different geographical locations, and climates. Seven common LC peaks that could be used for fingerprinting this common popular traditional Chinese medicine were found, and six were identified as substituted resinols (4 compounds), geniposidic acid and chlorogenic acid by LC-MS. Principal components analysis (PCA) indicated that samples from the Sichuan, Hubei, Shanxi and Anhui—the SHSA provinces, clustered together. The other objects from the four provinces, Guizhou, Jiangxi, Gansu and Henan, were discriminated and widely scattered on the biplot in four province clusters. The SHSA provinces are geographically close together while the others are spread out. Thus, such results suggested that the composition of the Eucommia Bark samples was dependent on their geographic location and environment. In general, the basis for discrimination on the PCA biplot from the original 46 objects× 7 variables data matrix was the same as that for the SHSA subset (36 × 7 matrix). The seven marker compound loading vectors grouped into three sets: (1) three closely correlating substituted resinol compounds and chlorogenic acid; (2) the fourth resinol compound identified by the OCH3 substituent in the R4 position, and an unknown compound; and (3) the geniposidic acid, which was independent of the set 1 variables, and which negatively correlated with the set 2 ones above. These observations from the PCA biplot were supported by hierarchical cluster analysis, and indicated that Eucommia Bark preparations may be successfully compared with the use of the HPLC responses from the seven marker compounds and chemometric methods such as PCA and the complementary hierarchical cluster analysis (HCA).

Identification and simultaneous analysis of harmane, harmol, isovitexin and vitexin in Passiflora incarnata extracts with a novel HLPC method

A high performance liquid chromatographic method for the simultaneous analysis of two flavonoids (iso-vitexin and vitexin), and three indole alkaloids (harmane, harmine, and harmol) was developed. This method was then utilised to quantitate levels of these five constituents in methanolic extracts of Australian Passiflora incarnata. HPLC analysis was performed using a Waters™ Novapak C18 (150 × 4 mm, 4 μm) column, with a gradient solvent system of methanol-water-acetic acid. Detection was achieved by PDA UV (254 nm) and fluorescence (excitation 254 nm, emission 414 nm), utilising the external standard method to obtain quantification.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

Raman microscopy of formamide-intercalated kaolinites treated by controlled-rate thermal analysis technology

Raman spectroscopy of formamide-intercalated kaolinites treated using controlled-rate thermal analysis technology (CRTA), allowing the separation of adsorbed formamide from intercalated formamide in formamide-intercalated kaolinites, is reported. The Raman spectra of the CRTA-treated formamide-intercalated kaolinites are significantly different from those of the intercalated kaolinites, which display a combination of both intercalated and adsorbed formamide. An intense band is observed at 3629 cm-1, attributed to the inner surface hydroxyls hydrogen bonded to the formamide. Broad bands are observed at 3600 and 3639 cm-1, assigned to the inner surface hydroxyls, which are hydrogen bonded to the adsorbed water molecules. The hydroxyl-stretching band of the inner hydroxyl is observed at 3621 cm-1 in the Raman spectra of the CRTA-treated formamide-intercalated kaolinites. The results of thermal analysis show that the amount of intercalated formamide between the kaolinite layers is independent of the presence of water. Significant differences are observed in the CO stretching region between the adsorbed and intercalated formamide.

Detailed analysis of a conservative difference approximation for the time fractional diffusion equation

Diffusion equations that use time fractional derivatives are attractive because they describe a wealth of problems involving non-Markovian Random walks. The time fractional diffusion equation (TFDE) is obtained from the standard diffusion equation by replacing the first-order time derivative with a fractional derivative of order α ∈ (0, 1). Developing numerical methods for solving fractional partial differential equations is a new research field and the theoretical analysis of the numerical methods associated with them is not fully developed. In this paper an explicit conservative difference approximation (ECDA) for TFDE is proposed. We give a detailed analysis for this ECDA and generate discrete models of random walk suitable for simulating random variables whose spatial probability density evolves in time according to this fractional diffusion equation. The stability and convergence of the ECDA for TFDE in a bounded domain are discussed. Finally, some numerical examples are presented to show the application of the present technique.