Multi-column Chromatography Research Articles

The impact of continuous multicolumn chromatography on biomanufacturing efficiency

Over the past few years, the capacity bottleneck in biomanufacturing has shifted from the upstream process to the purification process. In particular, capture chromatography steps have become a limiting factor in facility throughput. This is a direct result of the discrepancy between expression levels, which have increased by a factor of ten, and the capacity of biochromatography media, which has at best doubled. Continuous countercurrent multicolumn chromatography is a promising approach to deal with the capacity bottleneck, particularly if designed in a single-use format. BioSMB™ technology is a disposable bioprocess technology that allows standard capture chromatography steps, such as Protein A capture, to be executed with much less installed chromatographic media using a multicolumn chromatography process. This allows an economically favorable, single-use option where none has previously been available. Various case studies have demonstrated the advantages of this technology for various separations. I...

Multifraction separation in countercurrent chromatography (MCSGP)

The multicolumn countercurrent solvent gradient purification (MCSGP) process is a continuous countercurrent multicolumn chromatography process capable of performing three fraction separations while applying a linear gradient of some modifier. This process can then be used either for the purification of a single species from a multicomponent mixture or to separate a three component mixture in one single operation. In this work, this process is extended to the separation of multifractions, in principle with no limitation. To achieve this goal the MCSGP standard process is extended by introducing one extra separation section per extra fraction to be isolated. Such an extra separation section is realized in this work through a single additional column, so that a n fraction MCSGP process can be realized using a minimum of n columns. Two separation processes were considered to experimentally demonstrate the possibility of realizing a four-fraction MCSGP unit able to purify two intermediate products in a given multicomponent mixture. The first one was a model mixture containing four different proteins. The two proteins eluting in the center of the chromatogram were purified with yields equal to 95% for the early eluting and 92% for the later eluting one. The corresponding purities were 94% and 97%, respectively. Such performance was well superior to that of the batch operation with the same modifier gradient which for the same purity values could not achieve yields larger than 67% and 81%, respectively. Similar performance improvements were found for the second separation where two out of seven charge variants which constitute the mAb Cetuximab currently available on the market have been purified in one single operation using a four-fraction MCSGP unit. In this case, yields of 81% and 65% were obtained with purities of 90% and 89%, respectively. These data compare well with the corresponding data from batch chromatography where with the same gradient and for the same purities, yield values not larger than 49% and 34%, respectively, could be achieved.

Chiral separation of systems of high eutectic composition by a combined process: Case study of serine enantiomers

A concept for the chiral resolution of racemic compounds by a combination of chiral pre-enrichment by continuous multi-column chromatography and final purification using selective crystallization is presented. The system of serine enantiomers in water represents a typical racemic compound-forming system with very high eutectic compositions in the chiral system, which is generally considered as unfavorable for selective crystallization. It is shown exemplarily, how the solvent and temperature-dependent formation of hydrates can be exploited to allow efficient chiral resolution by a combined process for this and similar systems.

Intermittent simulated moving bed chromatography: 3. Separation of Tröger's base enantiomers under nonlinear conditions

One of the modified simulated moving bed (SMB) processes, the intermittent SMB (I-SMB) process, has been recently analyzed theoretically [1] and its superior performance compared to the conventional SMB process has been demonstrated at a rather low total feed concentration through experiments and simulations [2]. This work shows that the I-SMB process outperforms the conventional SMB process also at high feed concentration where the species are clearly subject to a nonlinear adsorption isotherm. In the case of the separation of the Tröger's base's enantiomers in ethanol on ChiralPak AD, the two processes operated in a six-column 1-2-2-1 configuration (one column in sections 1 and 4 and two columns in sections 2 and 3) and in a four-column 1-1-1-1 configuration (one column in each section) are compared at high feed concentration through both experiments and simulations. Even under nonlinear conditions the four column I-SMB process can successfully separate the two enantiomers achieving purity levels as high as the two six column processes and exhibiting better productivity.

Experimental assessment of simulated moving bed and varicol processes using a single-column setup

A novel single-column setup for experimentally reproducing the steady periodic behavior of simulated countercurrent multicolumn chromatography is presented. The system relies on accurate online monitoring of the outlet effluent composition, processing the measured data through a node balance, and feeding it back into the column with an appropriate time delay using a multi-pump configuration to reproduce the desired inlet stream. The feasibility of the proposed system is demonstrated on the linear separation of two nucleosides using three different column configurations, which include both synchronous and asynchronous port switchings. By judiciously selecting the switching interval for process startup and applying a model-based startup procedure, the periodic state can be attained in just one or two cycles. Therefore, mobile phase and solute consumptions required to experimentally reproduce the periodic state of the equivalent multicolumn process are reduced to a minimum. This may be an economic, optimal manner of experimentally testing a set of operating conditions or cycle policy to achieve a given separation performance for a new multicolumn chromatographic separation.

Automated chromatographic system for the simultaneous measurement of plasma pregnenolone and 17-hydroxypregnenolone by radioimmunoassay

A new, simple, rapid and highly practicable automated chromatographic system for the separation, and a sensitive radioimmunoassay system for the subsequent measurement of pregnenolone and 17-hydroxypregnenolone has been developed. Pregnenolone and 17-hydroxypregnenolone were extracted with methylene chloride and separated from cross-reacting steroids by mechanised Sephadex-LH20 multi-column chromatography. Anti-pregnenolone and anti-17-hydroxypregnenolone were obtained by immunising rabbits with pregnenolone-20-oxime-BSA and 17-hydroxypregnenolone-20-oxime-BSA. The lower detection limit of the assay is 0.15 and 0.28 nmol/l for pregnenolone and 17-hydroxypregnenolone, respectively. Normal values for this assay in young male adults, in adult females, and in prepubertal boys and girls were established as a basis for the functional diagnosis of androgen excess syndromes/steroidogenesis defects.

Increased Flexibility in Multicolumn Chromatography (MC[sbnd]HPLC) via On-line Effluent Mixing Technique

Abstract In continuation of our work dealing with multicolumn HPLC (MC[sbnd]HPLC) we describe in this paper an on-line on-column fraction trapping technique based on effluent mixing. To a normal two-column switching set-up (in this case with two RP columns) an additional high-pressure pump gets inserted into the connection line between column A and column B via a low dead volume mixing tee. The in-line respectively off-line switching of pump B and the mobile phase B is time controlled by using a high pressure switching valve. With this set-up it is possible to mix on-line an effluent fraction from column A and transferred onto column B with a highly polar and pH-controlled (e.g. aqueous buffer) new effluent, to reduce or adjust significantly the overall elution strength of this mixed transferred solvent. Thus, several chromatographically effective possibilities can be created in a simple manner, which are for example: (a) pronounced peak compression respectively on-column concentration on column B; (b) du...

Celite multi-column chromatography for the simultaneous separation of progesterone, deoxycorticosterone and 17α-hydroxyprogesterone from small plasma or tissue samples

A rapid, inexpensive and reliable chromatographic method has been developed for the simultaneous separation of progesterone, deoxycorticosterone and 17α-hydroxyprogesterone on 15-cm Celite columns using n-heptane—benzene—methanol—water (100:35:80:20) as mobile phase. Six columns can be run in parallel within an hour with the assistance of nitrogen pressure. There is negligible column-to-column or batch-to-batch variation with consistent high recoveries. Columns are easily and quickly prepared and being inexpensive are disposable which is important when working with radioactivity. Using this method three important corticosteroids can be rapidly and reliably isolated from 20–30 small plasma or tissue samples per day.

Longitudinal Studies of Plasma Aldosterone, Corticosterone, Deoxycorticosterone, Progesterone, 17– Hydroxyprogesterone, Cortisol, and Cortisone Determined Simultaneously in Mother and Child at Birth and during the Early Neonatal Period. I. Spontaneous Delivery*

In order to obtain the still lacking reference data of individual plasma steroids in the immediate postnatal period needed for the assessment of adrenocortical function in various neonatal maladaptation syndromes, aldosterone (A), corticosterone, deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol, and cortisone were simultaneously followed in the same human newborn in a single 250-500 microliters peripheral plasma sample obtained at constant times during the first week of life using a mechanized Sephadex LH-20 multicolumn chromatography and standardized RIAs. Mean concentrations in 12 spontaneously delivered full term newborns of either sex and in paired umbilical (UV) and peripheral maternal (MV) venous plasma are given in the table. Besides significant maternoumbilical gradients in each steroid, DOC, P, 17-OHP, and cortisone, originating predominantly from the fetoplacental unit, disappear rapidly with steadily increasing half-lives. A, corticosterone, and cortisol, however, remain elevated in comparison with later infancy, with the exception of a marked "glucocorticoid dip" in cortisol and corticosterone levels between 2 and 12 h after birth.

Fast liquid-dispensing device for multi-column liquid chromatography

Fast liquid-dispensing device for multi-column liquid chromatography

45: Longitudinal Study of 7 Plasma Corticosteroids (CS) in Mother and Child at Birth and during the Neonatal Period

The adrenal cortex seems to play an important role in the newborn's adaptation to extrauterine life. Since systematic studies on multiple CS in the same human newborn have not yet been reported, progesterone (P), deoxycorticosterone (DOC), 17α-hydroxyprogesterone (17α), corticosterone (B), aldosterone (A), cortisone (E) and cortisol (F) were followed in a single 250 μl peripheral plasma sample in 12 vaginally delivered full-term infants of either sex. Mean concentrations (after automated LH-20 multi-column chromatography and specific radioimmunoassays) were in ng/ml: Besides marked materno-fetal CS differences, P, 17α and DOC (predominantly of placental origin) disappear rapidly with varying half lives. A, B, E and F, however, remain elevated in comparison with later infancy, reflecting high adrenal biosynthetic activity. *(Approved by parents and Hospital's Ethical Committee)

Multi-column liquid chromatography : III. New techniques for multi-column liquid chromatography

New techniques for multi-column chromatography include a new system using nitrogen pressure and a system using a single pump to drive an eluent simultaneously through a number of chromatographic columns.

SIMULTANEOUS DETERMINATION OF SIX PLASMA CORTICOSTEROIDS IN PUPPIES DURING STANDARDIZED HAEMORRHAGIC SHOCK

11 puppies of 2–3 weeks of age and either sex were subjected to a standardized haemorrhagic shock. Aldosterone (A), corticosterone (B), cortisol (F), deoxycorticosterone (DOC), progesterone (P) and 17 α-OH-progesterone (17 α) were determined simultaneously in 250 ul of peripheral plasma taken at constant dates before, during and after blood withdrawal and re-infusion. Our method included CH2Cl2 extraction, separation by automated Sephadex LH-20 multi-column chromatography and quantitation of each steroid by radioimmunoassay. During hypovolaemia, plasma levels of the main mineralocorticoid (A) and its precursors (P,DOC,B) increase much more than those of the main glucocorticoid (F) and its precursor (17α). These and additional data on electrolyte and glucose levels reflect the key role of the adrenal cortex in maintaining homoeostasis during shock in the early life period.

High-capacity multi-channel computerized read-out system for multi-column chromatography

A computerized multi-channel read-out system for multi-column chromatography is described. It has a capacity of a hundred chromatograms per day when three channels are used and this can be expanded to four hundred chromatograms per day by the use of all twelve channels. The system is compared with other high-capacity systems for automated chromatography.