Anything but Conventional Chromatography Approaches in Bioseparation.

While packed bed chromatography, known as conventional chromatography, has been serving the biopharmaceutical industry for decades as the bioseparation method of choice, alternative approaches are likely to take an increasing leading role in the next few years. The high number of new biological drugs under development, and the need to make biopharmaceuticals widely accessible, has been driving the academia and industry in the quest of anything but conventional chromatography approaches. In this perspective paper, these alternative approaches are discussed in view of current and future challenges in the downstream processing field.

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.

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.

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.

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.

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%).

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 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.

Editorial: ECAB focus issue: Engineered catalysts, robust, cost-effective and integrated bioprocesses and high-throughput screening.

How can technology and industrial biotechnology contribute to a more bio-based economy? At the 3 European Congress of Applied Biotechnology (ECAB3) in Nice in 2015, relevant topics and technologies and their contribution to sustainability were presented and discussed. In this issue of Biotechnology Journal, five special articles are selected from this conference, highlighting processes and technologies envisaging the development of engineered catalysts, robust bioprocesses, as well as high-throughput screening methods for process development.

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.

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).

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.

An extracellular polymer at the interface of magnetic bioseparations.

FucoPol, a fucose-containing extracellular polysaccharide (EPS) produced by bacterium Enterobacter A47 using glycerol as the carbon source, was employed as a coating material for magnetic particles (MPs), which were subsequently functionalized with an artificial ligand for the capture of antibodies. The performance of the modified MPs (MP-EPS-22/8) for antibody purification was investigated using direct magnetic separation alone or combined with an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and dextran. In direct magnetic capturing, and using pure protein solutions of human immunoglobulin G (hIgG) and bovine serum albumin (BSA), MP-EPS-22/8 bound 120 mg hIgG g(-1) MPs, whereas with BSA only 10 ± 2 mg BSA g(-1) MPs was achieved.

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.

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.

Magnetic aqueous two phase fishing: a hybrid process technology for antibody purification.

The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions.

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.

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.

Boronic acid-modified magnetic materials for antibody purification.

Aminophenyl boronic acids can form reversible covalent ester interactions with cis-diol-containing molecules, serving as a selective tool for binding glycoproteins as antibody molecules that possess oligosaccharides in both the Fv and Fc regions. In this study, amino phenyl boronic acid (APBA) magnetic particles (MPs) were applied for the magnetic separation of antibody molecules. Iron oxide MPs were firstly coated with dextran to avoid non-specific binding and then with 3-glycidyloxypropyl trimethoxysilane to allow further covalent coupling of APBA (APBA_MP).

Amino acid/water interactions study: a new amino acid scale.

Partition ratios of 8 free l-amino acids (Gln, Glu, His, Lys, Met, Ser, Thr, and Tyr) were measured in 10 different polymer/polymer aqueous two-phase systems containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.

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.

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.

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.

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.