Perfusion chromatography: an emergent technique for the analysis of food proteins

Perfusion chromatography is a technique arised to overcome the problem associated with mass transfer in the separation of large molecules such as proteins by high-performance liquid chromatography (HPLC). Perfusion media are constituted by two set of pores: throughpores (6000–8000 Å) and diffusive pores (800–1500 Å) which enable better access of macromolecules to the inner of the particle by the combination of convective and diffusive flow. As a consequence, times required for a chromatographic separation are reduced. Perfusion media are available in different chromatographic modes: reversed-phase, ion-exchange, hydrophobic interaction, and affinity. From the theoretical models developed to explain the dynamic of retention of solutes in perfusive supports, it was derived that efficiency of a separation was independent of the flow-rate and only depended slightly on the particle diameter. Furthermore, loading capacity was also independent of the superficial velocity. All these advantages have promoted the use of this chromatographic technique for the separation of biomolecules both in analytical and preparative chromatography. Characteristics of perfusion chromatography make this technique very interesting for the analysis of food proteins. Perfusion chromatography enables the assessment of protein composition of a foodstuff at sufficient speed and low cost to be suitable in routine analysis.     

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Monoliths have consolidated their position in bioseparation. More than 200 different applications have been reported in the past two decades and their advantages compared to conventional chromatography demonstrated. These include the high mass transfer efficiency due to the convective flow enabled by the macroporous character of the matrix. Recently plasmid DNA and viruses were separated with high efficiency and cryogels and monolithic superporous agarose were developed for capture of proteins from crude homogenates and separation of microorganisms or lymphocytes. Currently four companies manufacture monoliths mainly for analytical applications although monoliths with a volume of 0.8 liter are commercially available and 8 L are available as prototypes. A book entitled "Monolithic materials: preparation, properties and applications" was published in 2003 and became standard reference of the status of this area. This review focuses on the progress in monoliths that goes beyond the scope of this reference book. Less progress has been made in the field of bioconversions in spite of the fact that monolithic supports exhibit better performance than beads in enzymatic processing of macromolecules. It appears that the scientific community has not yet realized that supports for these applications are readily available. In addition, monoliths will further substantially advance bioseparations of both small and large molecules in the future.     

An Introduction to Monolithic Disks as Stationary Phases for High Performance Biochromatography

Monolithic stationary phases have revolutionized protein chromatography because they combine speed, capacity, and resolution in a unique manner. Since such stationary phases contain no particles but only flow‐through pores, the usual mass transfer restrictions to the chromatography of large molecules are not observed and extremely fast separations become possible. Recently the area of application of monolith chromatography has been extended to the separation and analysis of small molecules and plasmid DNA. This review summarizes the state of art in high performance monolith and especially high performance monolithic disk chromatography (HPMDC). The current understanding of the theory of protein HPMDC is summarized, while an introduction to the evolving field of small molecule HPMDC is attempted. The basic differences between the monolithic disks and columns packed with conventional stationary phases (including perfusion and micropellicular particles) but also monolithic columns (porous rods) are outlined. Finally, the potential of HPMDC to analytical and preparative biochromatography is demonstrated by a discussion of recent applications of chromatographic disks for protein isolation and bioprocess analysis.     

Use of monolithic supports in proteomics technology

An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC-MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC-MS/MS protein identification will be discussed.     

Protein adsorption and transport in polymer-functionalized ion-exchangers

A wide variety of stationary phases is available for use in preparative chromatography of proteins, covering different base matrices, pore structures and modes of chromatography. There has recently been significant growth in the number of such materials in which the base matrix is derivatized to add a covalently attached or grafted polymer layer or, in some cases, a hydrogel that fills the pore space. This review summarizes the main structural and functional features of ion exchangers of this kind, which represent the largest class of such materials. Although the adsorption and transport properties may generally be used operationally and modeled phenomenologically using the same methods as are used for proteins in conventional media, there are noteworthy mechanistic differences in protein behavior in these adsorbents. A fundamental difference in protein retention is that it may be portrayed as partitioning into a three-dimensional polymer phase rather than adsorption at an extended two-dimensional surface, as applies in more conventional media. Beyond this partitioning behavior, however, the polymer-functionalized media often display rapid intraparticle transport that, while qualitatively comparable to that in conventional media, is sufficiently rapid quantitatively under certain conditions that it can lead to clear benefits in key measures of performance such as the dynamic binding capacity. Although possible mechanistic bases for the retention and transport properties are discussed, appreciable areas of uncertainty make detailed mechanistic modeling very challenging, and more detailed experimental characterization is likely to be more productive.     

Applications of polymethacrylate-based monoliths in high-performance liquid chromatography

Monolithic columns were introduced in the early 1990s and have become increasingly popular as efficient stationary phases for most of the important chromatographic separation modes. Monoliths are functionally distinct from porous particle-based media in their reliance on convective mass transport. This makes resolution and capacity independent of flow rate. Monoliths also lack a void volume. This eliminates eddy dispersion and permits high-resolution separations with extremely short flow paths. The analytical value of these features is the subject of recent reviews. Nowadays, among other types of rigid macroporous monoliths, the polymethacrylate-based materials are the largest and most examined class of these sorbents. In this review, the applications of polymethacrylate-based monolithic columns are summarized for the separation, purification and analysis of low and high molecular mass compounds in the different HPLC formats, including micro- and large-scale HPLC modes.     

Generic method for systematic phase selection and method development of biochromatographic processes. Part I. Selection of a suitable cation-exchanger for the purification of a pharmaceutical protein

Even if the first protein therapeutics are now for more than 20 years on the market the selection of suitable adsorbents for the preparative downstream processing (DSP) of these biomolecules as well as the method development towards process conditions are still based mainly on 'trial and error'. Therefore, theses processes are not perfectly efficient, but indeed very time consuming and laborious. In this study a novel systematic method is introduced to find a suitable adsorbent (not necessarily the best one) with appropriate separation parameters for a specific separation with reduced effort. Following this strategy, the adsorbents must first be packed into columns under preparative conditions and then characterized completely with regard to, e.g. pressure drop, k'-values, plate heights (HETP curves), selectivity and capacity by using test substances, which are similar in their characteristics (molecular mass, size, charge distribution, hydrophobicity) to the target proteins. With the database once determined, a preselection of most suitable adsorbents including separation parameters is made regarding chromatographic and also economical properties. After this, preparative experiments must be conducted with a reduced number of adsorbents to figure out the individual influence of side components. This approach is demonstrated for the separation of an exemplary industrial protein mixture using cation-exchange chromatography (CEX). Characterization of different weak CEX-adsorbents is illustrated. After comparing these phases with each other, a first preselection and a prediction of suitable adsorbents is made. In the following preparative separation conditions (load, velocity, gradient) are determined for the preparative separations using the database and results of some additional experiments. The final comparison of separation performance in preparative scale confirms this selection and so the applicability of the new method.     

Characterisation of metal-chelate methacrylate monoliths

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.     

Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far—ammodytoxins (Atxs)—are contributing to the venom’s toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.     

Physicochemical Principles and Applications of Supercritical Fluid Chromatography (SFC). New analytical methods (19)

In supercritical fluid chromatography (SFC) compressed gases in the region of their critical temperature are used as mobile phases. SFC has important advantages over gas chromatography (GC) for the separation of low-volatile or thermally unstable substances. Like high pressure liquid chromatography (HPLC) and gel chromatography, it is used for various special applications and preparative separations, e.g. in the petroleum industry and in the separation of oligomers. SFC is of great interest in fundamental research on fluid extraction and for the determination of the physicochemical properties of fluid systems. In this contribution the most important physicochemical, methodological, and instrumental principles of SFC are summarized; characteristic physicochemical applications are the determination of capacity ratios, partition coefficients, partial molar volumes, interaction second virial coefficients, and difusion coefficients.     

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode.

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.     

Protein Separation with Flow-Through Chromatography

Proteins are isolated in the chemical process industries for a wide variety of uses. Isolation and separation are often conducted with chromatography. Conventional chromatography of proteins is often tedious and can suffer from poor efficiency and resolution. There is a well-known tradeoff between resolution and speed', Newer methods of protein chromatography seek to diminish the shortcomings of conventional methods. New methods are presently being investigated for process, preparative, and analytical applications.     

Protein separation using membrane chromatography: opportunities and challenges

Some of the problems associated with packed bed chromatography can be overcome by using synthetic macroporous and microporous membranes as chromatographic media. This paper reviews the current state of development in the area of membrane chromatographic separation of proteins. The transport phenomenon of membrane chromatography is briefly discussed and work done in this area is reviewed. The various separation chemistries which have been utilised for protein separation, along with different applications, are also reviewed. The technical challenges facing membrane chromatography are highlighted and the scope for future work is discussed.     

Coupling HPLC to GC: why? How? With what instrumentation?

Coupling LC to GC alleviates sample preparation in the sense of preseparation, cleanup, or enrichment and replaces conventional methods such as column liquid chromatography, enrichment by or filtration through sample preparation tubes, preparative thin-layer chromatography, or liquid-liquid partitioning. LC is more efficient in separation power, more rapid, and allows fully automatic integration of sample preparation into GC. Advantages are discussed for selected applications. The transfer techniques, as well as some key requirements for an LC-GC instrument, are briefly summarized.     

Mass transfer characteristics of plasmids in monoliths

The hydrodynamic properties and pore-structure of monoliths based on functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) were characterised by pulse response experiments using different probes representing a wide range of molecular mass. On a small scale, band spreading was found to be caused to the extent of more than 90% by extra-column effects. These monoliths have large channel diameters, providing a suitable chromatography adsorbent for processing of large molecules. Dynamic and static binding capacity for plasmid DNA was investigated. For our model plasmid, consisting of 4.9 kbp, a capacity of 7 mg/mL was observed in comparison to 0.3 mg/mL for a conventional medium designed for protein separation. When plasmids were loaded on the monolith a gradual increase in pressure drop was observed. The channels filled up and the cross-sectional area available for liquid flow decreased. Therefore, a higher pressure drop was observed during elution. This is caused by (i) shrinking of the channels as effect of the high salt concentration, (ii) high viscosity of the mobile phase due to high concentration of plasmids, and (iii) an increase of the hydrodynamic radius of the plasmid with salt concentration from 45 nm at 150 mM to 70 nm at 2 M NaCl, as measured by dynamic light scattering. These types of monoliths are considered to be the preferred adsorbents for plasmid separation.     

Carbon nanotube based stationary phases for microchip chromatography

The objective of this article is to provide an overview and critical evaluation of the use of carbon nanotubes and related carbon-based nanomaterials for microchip chromatography. The unique properties of carbon nanotubes, such as a very high surface area and intriguing adsorptive behaviour, have already been demonstrated in more classical formats, for improved separation performance in gas and liquid chromatography, and for unique applications in solid phase extraction. Carbon nanotubes are now also entering the field of microfluidics, where there is a large potential to be able to provide integrated, tailor-made nanotube columns by means of catalytic growth of the nanotubes inside the fluidic channels. An evaluation of the different implementations of carbon nanotubes and related carbon-based nanomaterials for microfluidic chromatography devices is given in terms of separation performance and ease of fabrication.     

Monolithic chromatography for elemental and speciation analysis

Monolithic supports are increasingly used in the field of chromatography. They are appropriate for different applications (e.g., separation of biomolecules, organic acids and inorganic anions). However, only a few research groups are investigating the potential of using monolithic phases for rapid separation of metal cations and elemental speciation analysis.Monolithic supports based on porous monolithic silica have been successfully applied in separation of alkaline-earth and transition-metal cations in environmental waters and high ionic-strength samples.The present review covers applications of monolithic supports for chromatographic separation of metal cations and the potential for using monolithic chromatography in elemental speciation analysis. We critically evaluate the performances and the advantages of monolithic supports and compare them to conventional particle-packed chromatographic supports.     

Application of pH-Zone-Refining Countercurrent Chromatography in the Preparative Separation of Alkaloids from Traditional Chinese Medicine

pH-Zone-refining countercurrent chromatography (CCC) is a recently developed new preparative separation method based on conventional CCC. The method uses a retainer base (or acid) in the stationary phase to retain the analyses in the column and an eluent acid (or base) to elute the analyses according to their pKa values and hydrophobicities. It produces a succession of highly concentrated rectangular peaks with minimum overlap similar to those observed in displacement chromatography. pH-zone-refining CCC has important advantages over the conventional CCC including an over 10-fold increase (up to 10 gram or more) in sample loading capacity, high concentration of fractions, and concentration of minor impurities. pH-zone-refining CCC has been successfully applied to the preparative separation of a variety of compounds including both acidic and basic derivatives of amino acid, hydroxyxanthene dyes, peptides, alkaloids, indole auxins, structural, geometrial and optical isomers.     

Functionalized cryogel monoliths for fast and selective separation of nucleic acids directly from crude lysate

The fast and selective separation of nucleic acids has been attractive recently because of their wide number of applications in the biomedical field such as the development of vaccines for infectious diseases, gene therapy, and diagnosis. Traditional approaches of nucleic acids separation are costlier, lengthy, and associated with possible denaturation because of the use of organic solvents in the elution step. Under this perspective, cryogels represent an attractive choice as a monolith stationary phase in column chromatography, which have proven efficient in recent chromatographic studies. Cryogels are the macroporous hydrogels with interconnecting properties between the pores. They allow the easy flow of large biomolecules with minimum mass transfer resistance. They are spongy in nature and possess good mechanical strength. Current article represents different developed functionalized cryogel monoliths for nucleic acids separation, their separation strategies, and challenges associated with further advancement in separation science.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.