Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial‐scale systems aiming at plasmid DNA purification.     

Purification and analysis of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography

We discuss the purification of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono–, di–, and tri–), mono‐PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS‐PAGE, dynamic light scattering, and SEC combined with multi‐angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono‐PEGylated HSA. A scaled‐down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.     

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.     

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%.     

Application of monoliths for plasmid DNA purification development and transfer to production

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.     

Hydrophobic interaction chromatography at low salt concentration for the capture of monoclonal antibodies

We evaluated hydrophobic interaction chromatography (HIC) at low salt concentration for the capture of proteins from feed stocks by using monoclonal antibodies as model samples. It was indicated that the HIC at low salt concentration on critical hydrophobicity supports has a potential for capturing hydrophobic monoclonal antibodies directly from large volumes of feed stocks and recovering bound monoclonal antibodies in high yield. On the other hand, the HIC at low salt concentration did not seem so useful for the capture of weakly hydrophobic monoclonal antibodies. The recovery of weakly hydrophobic monoclonal antibodies from columns packed with critical hydrophobicity supports was not quantitative and significantly decreased as the residence time of the monoclonal antibodies in the columns became longer.     

Size exclusion chromatography of plasmid DNA isoforms

There is considerable interest in using size exclusion chromatography (SEC) to analyze and purify specific plasmid isoforms, but there is currently no fundamental understanding of the effects of plasmid size and morphology on plasmid behavior in SEC. Experiments were performed for plasmids from 3.0 to 17.0 kbp in size. The linear and open-circular isoforms were generated from the supercoiled plasmid by appropriate enzymatic digestion. SEC retention data were obtained using a Sephacryl S-1000 SF resin packed column and an Agilent HPLC system over a range of flow rates using buffers of different ionic strength and composition. The plasmid partition coefficients, K_P, were evaluated from the first statistical moment of the chromatographic peak. The partition coefficient decreased with increasing plasmid size as expected; K_P varied from 0.299 to 0.045 for supercoiled plasmids of 3.0 to 17.0 kbp. The partition coefficient also increased with increasing ionic strength due to the compaction of the DNA associated with the shielding of the intramolecular electrostatic interactions. For any plasmid size, the supercoiled isoform had the highest K_P followed by the open-circular and then the linear isoform, consistent with independent estimates of the plasmid radius of gyration as determined by static light scattering. The experimental data were analyzed using available theoretical models for the partitioning of linear and cyclic polymer chains in well-defined pore geometries. These results provide important insights into the behavior of different plasmid isoforms in size exclusion chromatography.     

Performance of hydrophobic interaction ligands for human membrane‐bound catechol‐O‐methyltransferase purification

Despite of membrane catechol‐O‐methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines’ O‐methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl‐, epoxy‐, and octyl‐Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH₂PO_4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1–0.5, 0.25–1, and 1% of Triton X‐100. On butyl media, a stepwise strategy using 375 and 0 mM NaH₂PO₄, followed by three elution steps at 0.25, 0.7 and 1% Triton X‐100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.     

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values. Despite relatively narrow release specifications, the performance of the adsorbent in a given purification process could still vary from lot to lot. In this case, an alternative use test (one that properly captures the lot to lot variability) may be required to determine an acceptable range of variability for a specific process. In this work, we describe the separation of therapeutic protein monomer and aggregate species using hydrophobic interaction chromatography, which is potentially sensitive to adsorbent lot variability. An alternative use test is formulated, which can be used to rapidly screen different adsorbent lots prior to implementation in a large-scale manufacturing process. In addition, the underlying mechanism responsible for the adsorbent lot variability, which was based upon differences in protein adsorption characteristics, was also investigated using both experimental and modeling approaches.     

Liquid chromatography purification and study of uridilylpolynucleotide-(5′P→O)-tyrosine phosphodiesterase

Summary LC has been used as a tool for studying uridilylpolynucleotide-(5′P→O)-phosphodiesterase—an enzyme which hydrolyses specifically the phosphodiester bond between picarnaviral RNA and viral protein VPg. According to various chromatographic data, the enzyme forms two types of complex with nucleic acids: weak ones which dissociate in 200 mM KCl, and others which are stable at concentrations up to 900 mM KCl. 2.5-3-fold (preparative) or 6-fold (normal scale) purification of the enzyme was obtained by size-exclusion chromatography (SEC). Cation-exchange separation (4-fold purification) was found to be more suitable as the second enzyme purification step than the earlier anionexchange method used. Three forms of enzyme activity were discovered by hydrophobic-interaction chromatography on the enzyme preparation obtained by SEC. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Hydrophobic interaction chromatography of proteins: Thermodynamic analysis of conformational changes

For BSA and β-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (ΔH_ads), entropy (ΔS_ads), free energy (ΔG_ads) and heat capacity (Δc_p,ads) changes associated with adsorption we evaluated chromatographic data by the non-linear van’t Hoff model. Additionally, we performed isothermal titration calorimetry (ITC) experiments. van’t Hoff analysis revealed that a temperature raise from 278 to 308 K increasingly favoured adsorption seen by a decrease of ΔG_ads from −12.9 to −20.5 kJ/mol for BSA and from −6.6 to −13.2 kJ/mol for β-lactoglobulin. Δc_p,ads values were positive at 1.2 m (NH₄)₂SO₄ and negative at 0.7 m (NH₄)₂SO₄. Positive Δc_p,ads values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH₄)₂SO₄ concentration. ITC measurements showed that ΔH_ads is dependent on surface coverage already at very low loadings. Discrepancies between ΔH_ads determined by van’t Hoff analysis and ITC were observed. We explain this with protein conformational changes upon adsorption which are not accounted for by van’t Hoff analysis.     

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography

Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography.     

Recombinant protein purification using gradient assisted simulated moving bed hydrophobic interaction chromatography. Part II: process design and experimental validation

In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated.     

Adsorptive membrane chromatography for purification of plasmid DNA

Adsorptive membranes were investigated for the downstream processing of plasmid DNA by quantifying both separation efficiencies and adsorption uptake with the anion-exchange membranes. Separation efficiencies of the 10-ml Mustang-Q were measured using pulses of 6.1-kilo base pair plasmid DNA and lysozyme tracers, and comparing the responses for both conventional and reverse-flow operation. The plasmid exhibited nearly 200 plates/cm, almost as high efficiency as the protein despite the large difference in size. This behavior contrasts strongly with typical behavior for spherical porous particle packings, which predicted large decreases in efficiency with increases in tracer size. Batch adsorption isotherms for the 6.1-kilo base pair plasmid on small sheets of anion-exchange membranes at various ionic strengths showed high capacities for very large biomolecules. The maximum binding capacity for the membrane unit was calculated as 10 mg plasmid/ml, an order of magnitude greater than typical values reported for porous beads.     

Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification

New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.     

Plasmid DNA processing for gene therapy and vaccination: Studies on the membrane sterilisation filtration step

Membrane filtration through 0.2 μm pores is typically the last operation in the production of pharmaceutical grade plasmid DNA. The membrane sterilisation of purified DNA solutions containing plasmids and bacterial artificial chromosomes (BAC) is investigated in this paper. A linear relationship between total DNA transmission and vector size was observed when filtering through 0.2 μm polyvinylidene difluoride (PVDF) membranes. The percentage of DNA transmission assessed spectrophotometrically varied from 98 to 13% for vector sizes ranging from 6 to 116 kb. There was no significant change in transmission during filtration when controlled flux was increased from 0.1 to 2.3 mL/min cm² or DNA concentration changed from 25 to 100 μg/mL. For vectors ≥20 kb; (i) the level of backbone breakage increased with molecular weight, flux and number of filtration passes; (ii) consecutive filtration experiments indicated that greater DNA loss occurred during the first pass of filtration; and (iii) the use of polyethersulfone (PES) membranes with asymmetrical pores improved DNA transmission and decreased DNA damage. The addition of 150 mM NaCl in the formulation buffer improved filtration transmission by 47 and 11% for the 72 and 116 kb vectors, respectively. Complexation with polyethylenimine (PEI) and a lipid–integrin binding peptide (LI) complex did not improve product transmission.     

Resolution enhancement in hydrophobic interaction chromatography via electrostatic interactions

In this work, a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bonding β-phenylethylamine as a functional ligand to silica. This stationary phase can separate proteins with similar hydrophobicity that traditional hydrophobic resins cannot. Hen egg white was separated to examine the selectivity. The results show that the introduced electrostatic interactions are an important factor for the resolution enhancement and the new resin could have important applications in separation and purification of biological macromolecules.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.