Continuous purification of a clotting factor IX concentrate and continuous regeneration by preparative annular chromatography

Preparative continuous annular chromatography, a method to separate proteins in a truly continuous manner, was investigated in an industrial environment. Plasma-derived clotting factor IX concentrate was used as model protein. Separation of vitronectin, a common impurity in commercial available factor IX concentrates, from factor IX was studied and compared to conventional packed bed chromatography in batch mode. As sorbent, Toyopearl DEAE 650M was used. Regeneration was performed simultaneously with the purification of factor IX in continuous mode. All required parameters applied for preparative annular chromatography such as feed flow-rate and elution flow-rate were first estimated from experiments on conventional batch columns. Then preparative annular chromatography and conventional packed beds were compared regarding enrichment, purity and productivity. Three different process scenarios, the optimal batch process,the preparative annular chromatography process and the batch process equivalent to the preparative annular chromatography process were investigated. The productivity of the optimal batch process was higher than that of the preparative annular chromatography and batch process equivalent to the preparative annular chromatography process. Therefore the throughput could not be increased by the use of the continuous chromatographic system.     

Fiber Bragg grating photoacoustic detector for liquid chromatography

Fiber Bragg Gratings (FBGs) are known to be sensitive acoustic transducers and have previously been used for the photoacoustic detection of small solid samples. Here, we demonstrate the use of an FBG as an on-line detector for liquid chromatography. The FBG was inserted into a silica capillary and the photoacoustic response from the effluent was generated by a 10 ns pulsed laser. The acoustic pulse was quantified by the FBG through a characteristic change in the reflection spectrum. Good repeatability and linear response were obtained over three orders of magnitude (R(2) > 0.99), and the limit of detection of Coumarin 440 was determined to be 5 microM. The technique was successfully coupled to high performance liquid chromatography and applied to on-line analysis of a three-compound solution. Photoacoustic detection in liquid chromatography using FBGs is a label-free method, which can be applied to the detection of any chromogenic compound irrespective of its fluorogenic properties. It is a simple, inexpensive, and inherently micron-sized technique, insensitive to electromagnetic interference.     

Scale-down of continuous protein purification by annular chromatography. Design parameters for the smallest unit

Theoretically, in fixed-bed chromatography an infinite separation time or volume is available. In annular chromatography the separation time is limited by the time required for a single rotation. In a chromatograph with a smaller annulus than the theoretical one, the equilibration zone will overtake the feed zone. The required separation time is determined by a number of steps such as equilibration, loading, washing, elution, regeneration and the retention of the solute. A mathematical model has been developed to estimate the minimum radius of an annular chromatograph. The minimal geometry of an annular chromatograph was calculated for a model system immunoglobulin and bovine serum albumin.     

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.     

Novel carbon-fibre electrochemical detector for use in liquid chromatography

The preparation and application of a novel carbon-fibre flow electrode used in liquid chromatography are reported. The electrode is simple and flexible, easy to prepare and to change and is stable in organic solvents. Heat sealing of the needle holes holding the carbon fibre in the polyethylene cell tube prevents the mobile phase from leaking through the holes. A stable and repeatable response has been obtained. The detection limit for 3,5-orcinol and 2,7-dihydroxynaphthalene was ca. 1 ng.     

Structural optimization of a chiral selector for use in preparative enantioselective chromatography

Utilizing the immobilized-target strategy, the structure of a proline-derived chiral stationary phase was optimized for use in the preparative chromatographic separation of the enantiomers of two chiral selectors used in commercial chiral stationary phases. In this study, various N-acylated proline anilides were prepared and chromatographed on the commercial Pirkle-1J and -Burke 2 chiral stationary phases. The analyte which displayed the greatest retention without sacrifice of enantioselectivity (the 3,5-dimethoxyanilide of N-undecenoyl proline) was chosen for incorporation into the preparative chiral stationary phase. Once prepared, this phase shows increased analyte retention and enantioselectivity comparable to that of earlier phases derived from 3,5-dimethyl anilides of proline. The increased retention allows one to use mobile phases in which the target analytes are more soluble, hence greatly facilitating an increase in the through-put of a column of a given size.     

Choice of procedures for preparative chromatography

Quantitative criteria, necessary and sufficient, were developed basing on the analysis of asymtotic solutions of border equations in elution sorption dynamics for realization of selectivity inversion for components in a chromatographic system due to the effect of kinetic selectivity. Analysis is suggested for experiments, where the new approach to realization of chromatographic processes using effect of kinetic selectivity allows optimization of preparative separation of biologically active substances. The approach suggested implies shortening of the experiment duration for separation processes, which can be crucial in most systems where components to be isolated are labile, or the process economics suffers considerably due to mobile phase, or energy consumption.     

Recycling in preparative liquid chromatography

Summary Chromatographic recycling is used to allow the scale up of an analytical separation made on a 7 mm ID microparticle GPC column to a multi-gram separation on a 25 mm ID preparative column, filled with conventional particle size GPC gel.     

Continuous separation of green fluorescent protein by annular chromatography

The concept of annular chromatography was tested by separation of a real protein solution used in biotechnology. Green fluorescent protein was expressed in S. cerevisiae and the extract was continuously separated by a pressurized annular chromatograph packed with a Superdex 200 prep grade size-exclusion chromatography medium. Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and fluorescent intensity. The continuous mode was compared to batchwise operation. Under the assumption that equivalent fractions are collected, both modes are comparable.     

Factors contributing to intrinsic loading capacity in silica-based packing materials for preparative anion-exchange protein chromatography

Properties of the matrix and stationary phase which affect the intrinsic loading capacity of silica-based packing materials for preparative anion-exchange chromatography of proteins were investigated. Polyethyleneimine-coated controlled porosity glass beads ranging from 100 to 2000 A in pore diameter were used to evaluate the effects of pore diameter and surface area. Protein binding was found to depend on accessible, rather than total, support surface area. Consequently, wide-pore, high surface area media provide maximum intrinsic loading capacity. Increasing the number of positively charged sites on the stationary phase by increased coating or by quaternization of amines increases hemoglobin-binding capacity.     

Laser-induced fluorescence detector for liquid chromatography: applications to protein analysis

A laser-induced fluorescence detector for liquid chromatography was developed. This detector was assessed by utilizing it in conjunction with gel filtration chromatography. Using the 488 nm line of an argon ion laser for excitation and monitoring the emitted fluorescence centering at 535 nm, the limit of detection of fluorescein was 580 fM. Bovine serum albumin labeled with fluorescein was detected at a concentration of 500 fM.     

Ultrahigh-speed,ultrahigh-resolution preparative separation of protein biopharmaceuticals using membrane chromatography

This paper discusses ultrahigh-speed, ultrahigh-resolution preparative protein separation using an in-house designed membrane chromatography device. The performance of the membrane chromatography device was systematically compared with an equivalent resin-packed preparative column. Experiments carried out using model proteins showed that membrane chromatography gave more than four times greater resolution than the preparative column, while at the same time being more than 19 times faster. Membrane chromatography was therefore a better option, not only in terms of higher productivity but also in terms of higher product purity. Membrane chromatography was also superior in terms of resolving and presenting tracer impurity peaks in the chromatogram. Experiments carried out using monoclonal antibody samples showed that membrane chromatography was suitable for ultrahigh speed, and ultrahigh resolution fractionation of charge variants. This paper highlights and explains the need for proper device design for enabling the use of membrane chromatography for the efficient purification of protein biopharmaceuticals.     

Peak doubling in preparative liquid chromatography

Summary Peak doublets in LC are ascribed to longitudinal column zones of different permeability leading to a double front in the column.     

Fiber optic Z-cell for CZE

Fiber optic Z-cell for CZE was designed, constructed, tested and compared with on-column detection. Ten times higher sensitivity for Z-cell in comparison with on-column detection was achieved as expected from optical pathlength ratio. Linear dynamic range was > 4 orders of magnitude for both cells.