Microfluidic platform for rapid screening of bacterial cell lysis.

Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency. This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.

Thermodynamics of the adsorption of monoclonal antibodies in phenylboronate chromatography: Affinity versus multimodal interactions.

The aim of this work was to investigate the complex phenomena underlying the adsorption of an anti-human IL-8 (anti-IL8) monoclonal antibody (mAb) to m-aminophenylboronate (m-APBA) by Flow Microcalorimetry (FMC) and to understand the role of non-specific interactions in the adsorption process. FMC was exploited as a dynamic on-line method to measure instantaneous heat energy transfers in order to understand the surface phenomena underlying mAb's adsorption towards the synthetic ligand m-APBA under different pH (7.5, 8.5, 9.0, 9.5 and 10.0) and salt concentrations (0 and 150 mM NaCl). Results showed that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven (ΔHads<0), as expected for the predominant reversible esterification reaction between boronates and cis-diols-containing molecules. For all the pH conditions studied, thermograms presented a first exothermic peak, characteristic of the reversible esterification reaction between mAb (pI≥9.3) and m-APBA (pKa = 8.8), except at pH 9.0 in the presence of 150 mM NaCl, for which the thermogram presented a first endothermic peak. The heat of adsorption (ΔHads) obtained at conditions where cis-diol interactions were predominant was approximately -243 ± 38 kJ/mol against -82 ± 14 kJ/mol (p-value < 0.05) obtained at pH 9.0 with 150 mM NaCl. The observed shift in the thermogram profile at pH 9.0, 150 mM NaCl, and the consequent decrease of 60-70% in ΔHads were indicative of the promotion of electrostatic interactions between the protein and the ligand. Overall, and whereas the binding of the PBA ligand to mAb molecules has been described for decades as being affinity-based, our study demonstrates the multimodal behaviour of this interaction and contributes towards the understanding of the adsorption thermodynamics.

Multiplexed microfluidic fluorescence immunoassay with photodiode array signal acquisition for sub-minute and point-of-need detection of mycotoxins.

Portable, rapid, cost effective and simple analytical tools are in increasing demand to facilitate the routine monitoring of target chemical/biological compounds at the point-of-need. Such devices are highly relevant within the context of food safety, particularly concerning the screening of highly toxic and strictly regulated mycotoxins. To achieve ultrarapid detection of mycotoxins, namely aflatoxin B1, ochratoxin A and deoxynivalenol, at the point-of-need, a novel multiplexed bead-based microfluidic competitive immunosensor, coupled with an array of a-Si:H thin-film photodiodes for integrated fluorescence signal acquisition, is reported. Simultaneously measuring the initial binding rate for each analyte of the sample under analysis against an internal reference, this device provided limits of detection below 1 ng mL-1 for all mycotoxins in a single-step assay and within 1 minute after mixing the sample under analysis with a fluorescent conjugate. The compatibility of the device with the analysis of mycotoxins spiked in corn samples was further demonstrated after performing a sample preparation procedure based on aqueous two-phase extraction. The short times of analysis and sensitivities in the low ng mL-1 range make these devices potentially competitive with the lateral flow devices that are currently the standard for this application. Furthermore, this device architecture and concept is amenable of being expanded to other analytes in food safety, biomedical and other applications.

Advances, challenges and opportunities for point-of-need screening of mycotoxins in foods and feeds.

The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains.

M13 bacteriophage purification using poly(ionic liquids) as alternative separation matrices.

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology.

A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems.

Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20µL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE.

A simple method for point-of-need extraction, concentration and rapid multi-mycotoxin immunodetection in feeds using aqueous two-phase systems.

The rapid detection of mycotoxins in feed samples is becoming an increasingly relevant challenge for the food production sector, in order to effectively enforce current regulations and assure food and feed safety. To achieve rapid mycotoxin detection, several biosensing strategies have been published, many reaching assay times of the order of a few minutes. However, the vast majority of these rely on sample preparation based on volatile organic solvents, often comprising complex multi-step procedures and devoid of clean-up and/or concentration effects. Here, a novel sample preparation methodology based on a green, non-toxic and inexpensive polyethylene glycol-sodium citrate aqueous two-phase system is reported, providing single-step extraction and concentration of three target mycotoxins within 20min: aflatoxin B1 (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON). With point-of-need applications in mind, the extraction procedure was optimized and validated using a rapid multi-toxin microfluidic competitive immunoassay. The assay was successfully tested with spiked complex solid matrices including corn, soy, chickpea and sunflower-based feeds and limits of detection of 4.6ngg-1±15.8%, 24.1ngg-1±8.1% and 129.7ngg-1±53.1% (±CV) were obtained in corn for AFB1, OTA and DON, respectively. These sensitivities are fit-for-purpose at the required regulatory and recommended limits for animal feed, providing an effective and safe semi-quantitative mycotoxin analysis that can be performed in the field.

Predicting protein partition coefficients in aqueous two phase system.

The present work aims to achieve an additional insight into the protein partitioning behavior in aqueous two phase systems (ATPSs), together with a study on the viability of a semi-empirical model based on continuum electrostatics to predict the protein partition characteristics. The partitioning behaviors of 14 globular proteins, with different properties, were explored in three polymer/polymer ATPSs. By the Collander equation, a linear correlation between protein partitioning coefficients in all systems was observed. Using the semi-empirical model it was possible to predict the partitioning behavior of proteins. The electrostatic energy depends on the protein size and ATPSs characteristics and varies in agreement with the difference in phase dielectric constants. Linear correlation of nonpolar energy, and the solvent accessible surface area was observed. Polymer structure and concentration have a significant influence on model viability. A good qualitative prediction of preferred phase for studied proteins was observed.

Miniaturization of aqueous two-phase extraction for biological applications: From micro-tubes to microchannels.

Aqueous two-phase extraction (ATPE) is a biocompatible liquid-liquid (L-L) separation technique that has been under research for several decades towards the purification of biomolecules, ranging from small metabolites to large animal cells. More recently, with the emergence of rapid-prototyping techniques for fabrication of microfluidic structures with intricate designs, ATPE gained an expanded range of applications utilizing physical phenomena occurring exclusively at the microscale. Today, research is being carried simultaneously in two different volume ranges, mL-scale (microtubes) and nL-scale (microchannels). The objective of this review is to give insight into the state of the art at both microtube and microchannel-scale and to analyze whether miniaturization is currently a competing or divergent technology in a field of applications including bioseparation, bioanalytics, enhanced fermentation processes, catalysis, high-throughput screening and physical/chemical compartmentalization. From our perspective, both approaches are worthy of investigation and, depending on the application, it is likely that either (i) one of the approaches will eventually become obsolete in particular research areas such as purification at the preparative scale or high-throughput screening applications; or (ii) both approaches will function as complementing techniques within the bioanalytics field.

Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats.

For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.

Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: development and optimization of a washing step.

In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries - 99% - and purifications - protein purity of 81% and a purification factor of 16 out of a maximum of 20 - were achieved using 100mM d-sorbitol in 10mM Tris-HCl as washing buffer and 0.5M d-sorbitol with 150mM NaCl in 10mM Tris-HCl as elution buffer. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.

Modeling the partitioning of amino acids in aqueous two phase systems.

A new model to obtain fast prediction of partition coefficients in polymer/polymer aqueous two phase systems (ATPSs) is presented, using amino acids as test systems. In particular, the partitioning behavior of eleven amino acids (glycine, alanine, leucine, phenylalanine, lysine, arginine, histidine, aspartic acid, glutamic acid, glutamine and serine) has been studied in 6 polymer/polymer ATPSs, formed by different pairs of nonionic polymers, including polyethylene glycol (PEG), Dextran, Ucon and Ficoll at 0.15M NaCl in 0.01M sodium phosphate buffer. The partition coefficients of the amino acids in the different ATPSs under study showed linear correlations as described by the Collander equation. Based on continuum electrostatics (CE), a semi-empirical model was developed to study the partitioning behavior in ATPSs. The approach employs a thermodynamic cycle where the electrostatic and nonpolar contributions to the free energy of partition are assumed to be additive. Three systems were chosen for the modeling studies: PEG-Dextran, PEG-Ficoll and Ficoll-Dextran. In general, the model was found to correctly predict the preferred phase for the studied amino acids, and, except for the charged ones, a good quantitative correlation of the calculated and experimental partition free energies was also obtained (e.g., with RMSE values of 150Jmol(-1) for PEG-Ficoll). The model performance could be improved by grouping amino acids according to their electrostatic properties, resulting in very good quantitative partition coefficient predictions (e.g., RMSE values for nonpolar amino acids of 29, 16 and 0.4Jmol(-1) for PEG-Dextran, PEG-Ficoll and Ficoll-Dextran system, respectively). The good performance of the proposed model in predicting partition coefficients of amino acids, the building blocks of proteins, offers a good prospect to its application to protein molecules and complexes.

Study of organic compounds-water interactions by partition in aqueous two-phase systems.

Partition coefficients of fourteen organic compounds were determined in 10 or 20 different polymer/polymer aqueous two-phase systems (ATPS) all at physiological pH (0.15M NaCl in 0.01M phosphate buffer, pH 7.4). Solute-specific coefficients characterizing different types of solute-water interactions for the compounds examined were determined by the multiple linear regression analysis. It is shown that (i) the partition behavior for the polar organic compounds is affected not only by dipole-dipole and hydrogen-bond interactions with aqueous environment but, notably, in most cases also by dipole-ion interactions; (ii) it is possible to predict partition behavior for compounds with pre-determined solute-specific coefficients in ATPS with characterized solvent features; and (iii) linear combinations of the solute-specific coefficients for the organic compounds might be useful in the development of quantitative structure-activity relationship (QSAR) analysis to describe their odor detection threshold.

Analysis of amino acid-water interactions by partitioning in aqueous two-phase systems. I--amino acids with non-polar side-chains.

Partition ratios of 10 L-amino acids with non-polar side chains (Gly, Ala, Val, nor-Val, Ile, Leu, nor-Leu, Phe, Trp and Pro) were measured in ten different polymer/polymer aqueous two-phase systems (ATPS) containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4. The solute-specific coefficients representing the solute dipole-dipole, hydrogen bonding and electrostatic interactions with aqueous environment for the compounds examined were determined by the multiple linear regression analysis using the modified linear solvation energy relationship equation. It is shown that linear combinations of these coefficients are correlated with the amino acid lipophilicity/hydrophobicity scales reported in the literature. The results obtained imply that the solute-specific coefficients may be used as solute descriptors for quantitative structure-property relationship (QSPR) analysis.

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: from concept to process.

An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.

Solvatochromic relationship: prediction of distribution of ionic solutes in aqueous two-phase systems.

Partition ratios of several ionic compounds in 20 different polymer/polymer aqueous two-phase systems (ATPS) containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4, were determined. The differences between the electrostatic properties of the phases in all the ATPS were estimated from partitioning of the homologous series of dinitrophenylated-amino acids. Also the solvatochromic solvent parameters characterizing the solvent dipolarity/polarizability (π*), solvent hydrogen-bond donor acidity (α), and solvent hydrogen-bond acceptor basicity (ß) of aqueous media were measured in the coexisting phases of the ATPS. The solute-specific coefficients for the compounds examined were determined by the multiple linear regression analysis using the modified linear solvation energy relationship equation. The minimal number of ATPS necessary for determination of the coefficients was established and 10 ATPS were selected as a reference ATPS set. The solute-specific coefficients values obtained with this reference set of ATPS were used to predict the partition ratios for the compounds in 10 ATPS not included in the reference set. The predicted partition ratios values were compared to those determined experimentally and found to be in good agreement. It is concluded that the presented model of solute-solvent interactions as the driving force for solute partitioning in polymer/polymer ATPS describes experimental observations with 90-95% accuracy.

Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography.

This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm(3) of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm(3) /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of pDNA was recovered in the flow through while 66-71% of impurities remained bound (~2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm(3) ) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm(3) of alkaline lysate per hour per dm(3) of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration.

Salt effects on solvent features of coexisting phases in aqueous polymer/polymer two-phase systems.

The solvatochromic parameters characterizing the solvent dipolarity/polarizability (π*), solvent hydrogen-bond donor acidity (α), and solvent hydrogen-bond acceptor basicity (ß) of aqueous media were measured in the coexisting phases of aqueous Dextran-Ficoll, Dextran-Ucon, Dextran-PEG, PEG-Ucon, Ficoll-Ucon, and Ficoll-PEG two-phase systems (ATPS). Ionic composition of each ATPS included 0.15M KCl, 0.15M KBr, 0.15M NaBr, 0.1M Na(2)SO(4), and 0.1M Li(2)SO(4) in 0.01 M sodium phosphate buffer (NaPB), pH 7.4; and 0.01 M and 0.11 M sodium phosphate buffer, pH 7.4. Partition ratios of sodium salts of dinitrophenylated (DNP) amino acids with aliphatic side-chains (glycine, alanine, norvaline, norleucine, and α-amino-n-caprylic acid) were measured in all ATPSs, and the results were evaluated in terms of the differences between the relative hydrophobicity (parameter E) and the electrostatic properties (parameter C) of the aqueous media of the coexisting phases. It was established that parameter E is described by a linear combination of the differences between the solvent dipolarity/polarizability (Δπ*) and between the solvent hydrogen-bond acidity (Δα) of the media in the coexisting phases. Parameter C depends on the phase forming polymer pair and is shown to be described by a linear combination of three parameters: the differences between the solvent hydrogen-bond acidity (Δα) and between the solvent hydrogen-bond basicity (Δß) of the media in the coexisting phases, and a measure of the effect of a given salt additive on the hydrogen bonds in water. This effect was represented by a parameter (K(b-l)), characterizing the equilibrium between populations of hydrogen bonds with a bent hydrogen bond conformation and with linear hydrogen bond conformation affected by a given salt additive.

Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads.

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.