A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process

Biomolecules are often purified via solvent gradient batch chromatography. Typically suitable smooth linear solvent gradients are applied to obtain the separation between the desired component and hundreds of impurities. The desired product is usually intermediate between weakly and strongly adsorbing impurities, and therefore a central cut is required to get the desired pure product. The stationary phases used for preparative and industrial separations have a low efficiency due to strong axial dispersion and strong mass transfer resistances. Therefore a satisfactory purification often cannot be achieved in a single chromatographic step. For large scale productions and for very valuable molecules, countercurrent operation such as the well known SMB process, is needed in order to increase separation efficiency, yield and productivity. In this work a novel multicolumn solvent gradient purification process (MCSGP-process) is introduced, which combines two chromatographic separation techniques, which are solvent gradient batch and continuous countercurrent SMB. The process consists of several chromatographic columns, which are switched in position opposite to the flow direction. Most of the columns are equipped with a gradient pump to adjust the modifier concentration at the column inlet. Some columns are interconnected, so that non pure product streams are internally, countercurrently recycled. Other columns are short circuited and operate in batch mode. As a working example the purification of an industrial stream containing 46% of the hormone Calcitonin is considered. It is found that for the required purity the MCSGP unit achieves a yield close to 100% compared to a maximum value of a single column batch chromatography of 66%.     

Column chromatographic separation of acidic and neutral and basic metabolites of tyrosine and dihydroxyphenylalanine

Liquid column chromatographic techniques for separation of tyrosine; dihydroxyphenylalanine; and acidic, neutral, and basic metabolites of tyrosine and catecholamines are described. These techniques permitted simultaneous separation of a wide spectrum of acidic and neutral metabolites of tyrosine and dihydroxyphenylalanine on one column. Rapid and complete separation of basic metabolites was carried out on a second column.     

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.     

Instrumentation for advanced microseparations in pharmaceutical analysis and proteomics

Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.     

Chemochromatography: its use for the separation of nikkomycins X and Z

A novel mode of reversed-phase high-performance liquid chromatography in which the mobile phase reacts chemically with the compounds to be separated was developed. Nikkomycin X and nikkomycin Z, two natural isomeric nucleoside peptide antibiotics, move as a single peak on a C18 reversed-phase column using an aqueous trifluoroacetic acid mobile phase. Addition of sodium bisulfite (1.0%) to the mobile phase results in the formation of a polar bisulfite addition product with nikkomycin X, but not with nikkomycin Z, inside the HPLC column. This type of reactive chromatography, or chemochromatography, led to the analytical and preparative separation of nikkomycins X and Z which are normally very intractable to separation by conventional chromatographic techniques.     

A rapid radiometric assay for adenosine deaminase

A rapid radiochemical procedure for the measurement of adenosine deaminase is described. The method employs phospho-Sephadex, a weak cation exchanger, which permits the enzymic product inosine to pass unretarded through the gel while the radioactive substrate adenosine is retained. Use of a Millipore filter manifold permits rapid processing of samples and eliminates time-consuming column chromatographic, electrophoretic, or paper chromatographic techniques required for separation of product and substrate.The activity of adenosine deaminase was examined in spleen cell preparations prepared from normal CBA mice. Excellent agreement of results was obtained when the radioactive method was compared with two other independent assay techniques.     

Alternatives to chromatographic separations

Up to now, the productivity of mammalian cell culture has been perceived as limiting the productivity of the industrial manufacture of therapeutic monoclonal antibodies. Dramatic improvements in cell culture performance have changed this picture, and the throughput of antibody purification processes is gaining increasing attention. Although chromatographic separations currently are the centerpiece of antibody purification, mostly due to their high resolving power, it becomes more and more apparent that there may be limitations at the very large scale. This review will discuss a number of alternatives to chromatographic antibody purification, with a particular emphasis on the ability to increase throughput and overcome traditional drawbacks of column chromatography. Specifically, precipitation, membrane chromatography, high-resolution ultrafiltration, crystallization, and high-pressure refolding will be evaluated as potential large scale unit operations for industrial antibody production.     

Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.     

Continuous annular chromatography

The principle of continuous annular chromatography (CAC) has been known for several decades. CAC is a continuous chromatographic mode, which lends itself to the separation of multi-component mixtures as well as of bi-component ones. In CAC, the mobile and stationary phases move in a crosscurrent fashion, which allows transformation of the typical one-dimensional batch column separation into a continuous two-dimensional one. With the exception of linear gradient elution, all chromatographic modes have at present been applied in CAC. This review focuses on the capacity of CAC for preparative bioseparation. The historical developments and the predecessors of modern CAC are briefly summarized. The state-of-the-art in the theoretical prediction and simulation of CAC separations is discussed, followed by an overview of current CAC instrumentation and example applications, especially for the isolation of proteins and other bio(macro)molecules. In this context, issues of scale up as well as method development and transfer from batch to continuous CAC columns are discussed using recent bioseparation efforts as pertinent examples.     

Size exclusion chromatography of cellulose in LiCl/N,N-dimethylacetamide

Size exclusion of cellulose in LiCl/N,N-dimethylacetamide has been used for the past 15 years, yet much of the current research is still devoted to fundamental studies, as many issues regarding column calibration, separation mechanisms and solution behavior have not been resolved yet. The paper reviews practical aspects of sample preparation and it is demonstrated that sample heating and several techniques to aid solvent exchange call for reevaluation. It is further shown that the use of internal standard may introduce minor improvements in repeatability. The commonly used column calibration procedures, chromatographic conditions and applications are also reviewed. Further research is needed to understand the mechanisms of separation, to optimize column calibration and to facilitate and optimize sample preparation.     

Efficient MILP formulations for the optimal synthesis of chromatographic protein purification processes

The use of recombinant proteins has increased greatly in recent years, as well as the techniques used for their purification. The selection of an efficient process to purify proteins is a major bottleneck found when trying to scale up results obtained in the laboratory to a large-scale industrial process. One of the main challenges in the synthesis of downstream purification stages in biotechnological processes is the appropriate selection and sequencing of chromatographic steps. The objective of this work is to develop mixed integer linear programming models for the synthesis of protein purification processes. Models for each chromatographic technique rely on physicochemical data of a protein mixture, which contains the desired product and provide information on its potential purification. Formulations that are based on convex hull representations are proposed to calculate the minimum number of steps from a set of chromatographic techniques that must achieve a specified purity level and alternatively to maximize purity for a given number of steps. The proposed models are tested in several examples with experimental data and present time reductions of up to three orders of magnitude when compared to big-M formulations.     

Drug analysis by direct liquid introduction micro liquid chromatography mass spectrometry

The analytical capabilities of a micro high performance liquid chromatograph interfaced to an unchanged quadrupole mass spectrometer are presented. Continuous monitoring of the total micro liquid chromatographic effluent allows full scan chemical ionization mass spectra of from one to five nanograms of drugs and their metabolites to be recorded. The interface is a simple, inexpensive device which can be assembled from commercially available components. An eight microliter per minute flow rate of the micro liquid chromatographic eluant allows separation and identification of biologically important substances not amenable to gas chromatography mass spectrometry techniques. The sensitivity of micro liquid chromatography mass spectrometry performed as described is comparable with gas chromatography mass spectrometry and is achieved by introducing the total micro liquid chromatographic effluent into the chemical ionization ion source of the mass spectrometer. Selected ion monitoring provides 20 pg detection limits of phenothiazine tranquilizers injected on column.     

Determination of epsilon (gamma-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation

A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the epsilon (gamma-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of epsilon (gamma-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the epsilon (gamma-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.     

Use of cyclodextrins as chiral selectors for direct resolution of the enantiomers of fluoxetine and its metabolite norfluoxetine by HPLC

The goal of this work is to investigate the direct chromatographic separation of the enantiomers of fluoxetine and its active metabolite norfluoxetine. The liquid chromatographic retention behavior of these enantiomers on a β-cyclodextrin bonded-phase column was investigated with respect to mobile phase composition, pH, ionic strength, and solvent selectivity. Relationships were established between these factors and the three most important chromatographic parameters: retention time, resolution, and selectivity. Most of the evidence suggests that the unique selectivity of this column isdue to inclusion complex formation, which provides the physical basis for enantiomeric resolution. After these studies a set of optimum chromatographic conditions was chosen for the simultaneous separation/determination of a mixture of the four enantiomers using fluorescence detector. © 1993 Wiley-Liss, Inc.     

Displacement effect during HPLC preparative purification of human insulin

HPLC plays a key role in the preparative purification of human insulin. A21-desamidoinsulin is one of the impurities that possesses the chromatographic behavior similar to that of insulin and hence separation from this by-product is rather difficult at the process scale. During the optimization of insulin reversed-phase HPLC purification, when a column was sufficiently overloaded, the effect of displacement of A21-desamidoinsulin molecules from active groups of sorbent by insulin ones was observed. It was suggested that monocarboxylic acid and organic modifier in mobile phase are responsible for the esterification during which the formed ester promotes the displacement effect. This effect was studied in order to optimize the purification of human insulin at the process scale.     

Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.     

Liquid chromatography of recombinant proteins and protein drugs

Many recombinant proteins (rPRTs) have a high bioactivity and some of them may eventually be classified as drugs beneficial to human health, recombinant human protein drugs (rPDs). rPDs are a high-technology product with all the associated economic benefits, therefore the liquid chromatography (LC) of rPRT is different from that of proteins isolated in laboratory scale for purely research purposes. The design of a purification scheme for an rPRT depends on the intended function of the purified rPRT, as a pure sample for research in small scale, or as a product for industrial production. This review paper mainly deals with the latter instance, producing rPD at a large scale. Pharmaceutical economics is considered not only for each step of purification, but also the whole production process. This strategy restricts the content of this review paper to the factors affecting the optimization source, the character of rPRT in up-stream technology and the purification of the rPRT in down-stream production. In the latter instance, the purification step is required to be as efficient as possible and LC is the core of the refined purification method, which is either a single LC method or combination of LC methods, sometimes, it may be a combination of LC and other non-LC separation methods comprising an optimized purification technology. Here some typical examples of rPRT purification at the large scale, recent developments, such as protein folding liquid chromatography, short column chromatography, and new packing material and column techniques are introduced.     

Protein/enzyme inactivation during different chromatographic methods of separation.

Protein denaturations encountered during the different types of chromatographic separations are presented. The analysis of different protein denaturations presented along with the causes of such denaturations provides a judicious framework to compare protein denaturations encountered by such separation techniques. Especially of interest are those studies which compare the mass recovery of proteins and the retention of activity by different chromatographic techniques. Reversed-phase chromatography is presented even though it is utilized nowadays only for specialized cases such as separation of small peptides. It appears that relatively mild interactions that are encountered generally in hydrocarbon-interaction chromatography are favorable to the preservation of the native (active) protein state. The few available mechanistic studies presented provide judicious physical insights into protein conformational behavior on chromatographic columns.     

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 μm). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.     

Predictability of Enantiomeric Chromatographic Behavior on Various Chiral Stationary Phases Using Typical Reversed Phase Modeling Software

Pharmaceutical companies worldwide tend to apply chiral chromatographic separation techniques in their mass production strategy rather than asymmetric synthesis. The present work aims to investigate the predictability of chromatographic behavior of enantiomers using DryLab HPLC method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. Three different types of chiral stationary phases were tested for predictability: macrocyclic antibiotics‐based columns (Chirobiotic V and T), polysaccharide‐based chiral column (Chiralpak AD‐RH), and protein‐based chiral column (Ultron ES‐OVM). Preliminary basic runs were implemented, then exported to DryLab after peak tracking was accomplished. Prediction of the effect of % organic mobile phase on separation was possible for separations on Chirobiotic V for several probes: racemic propranolol with 97.80% accuracy; mixture of racemates of propranolol and terbutaline sulphate, as well as, racemates of propranolol and salbutamol sulphate with average 90.46% accuracy for the effect of percent organic mobile phase and average 98.39% for the effect of pH; and racemic warfarin with 93.45% accuracy for the effect of percent organic mobile phase and average 99.64% for the effect of pH. It can be concluded that Chirobiotic V reversed phase retention mechanism follows the solvophobic theory. Chirality 25:506–513, 2013. © 2013 Wiley Periodicals, Inc.