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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 (ΔH ads <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 (ΔH ads ) 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 ΔH ads 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. [ABSTRACT FROM AUTHOR] more...

This work addresses the feasibility of scaling-up phenylboronic acid (PBA) chromatography for the purification of human mAbs directly from Chinese Hamster Ovary cell culture supernatants. Column performance optimization regarding superficial velocity and feed volume was performed with the best results being achieved with superficial velocities of 5.1, 15.3 and 25 cm/min in the adsorption, wash and elution steps, respectively. The column volume was scale-up 100 times from 0.4 cm 3 (laboratory scale) to 4, 16 and 40 cm 3 (preparative scale). This 100-fold scale-up was successfully achieved with a recovery yield of 97.7% and a protein purity of 82.6%. Following purification, purified IgG fractions were characterized in terms of isoelectric point, size, CHO proteins and genomic DNA content. Overall results suggest that using ProSep®-PB for mAbs purification is simple, reproducible, robust and scalable without compromising the target molecule integrity and purity. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

Abstract: 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 MgCl2 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 1.7M and 1.0M, 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. [Copyright &y& Elsevier] more...

Abstract: The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6±3.1% of RNA, 94.6±0.8% of proteins and 96.7±11.7% of gDNA were cleared after 30min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2±4.0%), even though the RNA (65.5±2.8%), protein (84.4±1.3%) and gDNA clearance (44.7±14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps. [Copyright &y& Elsevier] more...

Highlights: [•] The partitioning of amino acids was studied in aqueous two phase systems (ATPS). [•] The Collander equation was successfully applied. [•] A semi-empirical model was developed to predict partition coefficients. [•] The model shows a good predicting capacity on amino acids partitioning. [ABSTRACT FROM AUTHOR] more...

The therapeutic use of proteins has created an increasing demand for feasible and economical methods for both up- and downstream processes. However, whereas upstream processes have attracted substantial investment and commercial attention, downstream processing has been overlooked, causing a production bottleneck that is shifting the costs of production. This review focuses on the use of aqueous two-phase extraction as an option for the downstream processing of therapeutic proteins. It is a potential and promising liquid–liquid extraction technique for the purification of biomolecules, such as monoclonal antibodies, growth factors and hormones, that combines a high selectivity and biocompatibility with an easy scale-up and continuous operation mode. [Copyright &y& Elsevier] more...