Multi-column Systems Research Articles

Bracing requirements for multiple parallel nonidentical columns with initial curvature

For a system consisting of multiple columns, horizontal discrete braces are often used to increase the system’s strength by decreasing the effective length of columns. Since the bracing requirements for such systems in current design standards were developed based on the assumption of identical columns with pin-ends, an analytical method is proposed to evaluate the ideal brace stiffness and brace forces for multiple columns by accounting for the stiffness interaction among columns and braces. For systems with uniform column lateral stiffness, the explicit solution for the maximum brace force is derived, and a simple formula is subsequently proposed by fitting the analytical results. The proposed formula explicitly accounts for the influences of column initial curvature and semi-rigid end connections on the maximum brace force. The method is comprehensive and applicable to multi-column systems with nonuniform column lateral stiffness, which can result from differences in column sizes, end connections, or applied loads. The results obtained from the proposed formulae are verified against those of finite element analyses. The ideal brace stiffness and brace forces for a system containing a distinctive column with a greater moment of inertia than the other columns are investigated. The results show that the location of the distinctive column affects the magnitudes of the ideal brace stiffness and the maximum brace force, which is yet to be considered in current design standards.

Control strategy for multi-column continuous periodic counter current chromatography subject to fluctuating inlet stream concentration

Fluctuations of the inlet feed stream concentration are a challenge in controlling continuous multi-column counter current chromatography systems with standard methods. We propose a new control strategy based on calculated product column breakthrough from UV sensor signals by neglecting an impurity baseline and instead using the impurity to product ratio. This calculation is independent of the inlet feed concentration. In-silico simulation showed that the proposed method can calculate the product column breakthrough perfectly even with fluctuating and highly unstable inlet feed concentration during a loading cycle. Applying the proposed method to control a three column periodic counter current chromatography process with fluctuating inlet feed concentration resulted in constant column loading in each cycle, while using the standard method failed to do so. Unavoidable band broadening caused by diffusion and dispersion has been identified as an inherent limiting factor for accurate calculation of column breakthrough comparing inlet and outlet UV signals. The proposed advanced calculations increase the robustness of periodic counter current chromatography and extend the capability to process unstable inlet streams.

Algorithms to optimize multi-column chromatographic separations of proteins

The goal of this work was to provide a technical solution for the automated optimization of multi-column systems for protein separation and fractionation. Both algorithm and a software that can be downloaded are provided. In this algorithm, the length and order of the individual column segments can be considered. Various solutions are provided by the algorithm, including i) to obtain uniform peak distribution, ii) to park the different species at the inlet of the individual column segments, and iii) to elute all species as a single peak.Two representative examples are presented, showing the possibility to obtain uniform selectivity between monoclonal antibody (mAb) sub-units, and the on-column fractioning of intact mAbs.

Chromatography bioseparation technologies and in-silico modelings for continuous production of biotherapeutics.

The potential of continuous bioprocessing is hindered by the bottlenecks of chromatography processing, which continues to be executed in batch mode. Highlighting the critical drawbacks of batch chromatography, this review underscores the transition that the industry has made by implementing continuous upstream process without devising a working model for downstream chromatography operations. Even though multitude of process development initiatives have commenced, the review emphasizes the first principle models of chromatography on which these initiatives are built. Various models of continuous chromatography, which are essential, but not limited to multi-column systems, employed to congeal a unified process are reviewed. Advancements made by several mechanistic models and simulations to maximize productivity and performance are described, in an attempt to provide the integral tools. The modeling tools can be used for development of a strong model based control strategy and can be embedded into the continuous chromatography framework. The review addresses the limitations and challenges of the current modeling methods for development of robust mechanistic modeling and efficient unit operation platform in continuous chromatography.

Force Measurements of the Flow Around Arrays of Three and Four Columns With Different Geometry Sections, Spacing Ratios, and Incidence Angles

Abstract An experimental campaign for the flow around a stationary array of three and four columns with low aspect ratio, H/L = 1.5, piercing the water free surface, was carried out in a towing tank. These numbers of columns correspond to typical multi-column offshore systems, such as semi-submersibles (SS), tension leg platforms (TLPs), and floating offshore wind turbines (FOWTs). Three parameters were investigated: the spacing ratio between column centers (from two up to four characteristic lengths), current incidence angles, and column section geometries (circular, square, and diamond). The Reynolds number of the experiments was 100,000. Forces were measured in each column using a three degrees-of-freedom load cell, and results of lift and drag forces were presented for each column separately and the whole system. The results of mean and standard deviation of forces were assessed using a statistical uncertainty analysis procedure for finite length measurements’ signals. This methodology not only assesses the quality of the experimental data but also facilitates validation of numerical tools. The objectives of the current work were therefore manifold: to better understand the influence of the relative position, shape, and incidence angle on multi-column offshore structures; to create a reliable database for computational fluid dynamics (CFD) validation; and to prepare the path to flow-induced motions (FIMs) experimental and numerical work of free-moving multi-column offshore systems.

Enhanced adsorption performance of oxidised coconut coir for removal of Cd(II) ions by multi-column arrangement in series.

Biosorption potential of oxidised coconut coir (OCC) for removal of Cd(II) was evaluated by multi-column arrangement by connecting three columns in series. Effect of flow rate at 5, 10 and 15mL/min was studied at 30mg/L initial Cd(II) concentration. The dynamic capacity of the system was found to be 321, 206 and 83mg/L for 5, 10 and 15mL/min flow rates, respectively, by applying the bed depth service time model. Biosorbent usage rates for single-column and multi-column systems were compared. Better utilisation of biosorbent was observed when the columns are connected in series at similar operating parameters. A simple acid-base regeneration procedure was found to be effective in desorbing/regenerating the cadmium bound biosorbent. Adsorption efficiency was found to decrease from 76.3% for the first cycle to 72.2% and 70.6% in the second and third cycles, respectively. Regeneration efficiencies were more than 94% up to 3 cycles. The study highlights the effectiveness of the multi-column system in biosorption against the conventional single-column system.

Integrated continuous biomanufacturing platform with ATF perfusion and one column chromatography operation for optimum resin utilization and productivity

ABSTRACTA new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.

High performance affinity chromatography and related separation methods for the analysis of biological and pharmaceutical agents.

The last few decades have witnessed the development of many high-performance separation methods that use biologically related binding agents. The combination of HPLC with these binding agents results in a technique known as high performance affinity chromatography (HPAC). This review will discuss the general principles of HPAC and related techniques, with an emphasis on their use for the analysis of biological compounds and pharmaceutical agents. Various types of binding agents for these methods will be considered, including antibodies, immunoglobulin-binding proteins, aptamers, enzymes, lectins, transport proteins, lipids, and carbohydrates. Formats that will be discussed for these methods range from the direct detection of an analyte to indirect detection based on chromatographic immunoassays, as well as schemes based on analyte extraction or depletion, post-column detection, and multi-column systems. The use of biological agents in HPLC for chiral separations will also be considered, along with the use of HPAC as a tool to screen or study biological interactions. Various examples will be presented to illustrate these approaches and their applications in fields such as biochemistry, clinical chemistry, and pharmaceutical research.

Continuous countercurrent tangential chromatography for mixed mode post-capture operations in monoclonal antibody purification

Continuous Countercurrent Tangential Chromatography (CCTC) has been shown to demonstrate significant advantages over column chromatography including higher productivity, lower operational pressure, disposable flow path, and lower resin use. Previous applications of CCTC have been limited to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest. In this present article, a CCTC system was designed and tested for a post-capture antibody purification step. Mixed mode cation exchange-hydrophobic interaction chromatography resins with two different particle sizes were used to reduce host cell protein (HCP), leached protein A, DNA, and aggregates from a mAb stream after a protein A operation. Product output from CCTC was obtained at a steady-state concentration in sharp contrast to the periodic output of product in multi-column systems. The results show up to 101g of mAb/L of resin/hr productivity, which is 10× higher than in a batch column. A 5% yield increase (95% with CCTC vs. 90% in batch column) resulted from optimizing elution pH within a narrow operational window (pH 4–4.5). Contaminant removal was found to be similar to conventional column performance. Data obtained with the smaller particle size resin showed faster binding kinetics leading to reduced CCTC system volume and increased productivity. Buffer and water usage were modeled to show potential for utilization of in-line mixing and buffer tank volume reduction. The experimental results were used to perform a scale up exercise that predicts a compact CCTC flow path for 500 and 2000L batches using commercially available membranes. These results demonstrate the potential of using CCTC for post-capture operations as an alternative to packed bed chromatography, and provide a framework for the design and development of an integrated continuous bioprocessing platform based on CCTC technology.

Designing affinity chromatographic processes for the capture of antibodies. Part I: A simplified approach

Protein A affinity chromatography is a standard technique for the purification of therapeutic antibodies. Recently, multi-column chromatographic processes have emerged to turn classical capture processes into more efficient and continuous systems. The design of such chromatographic processes, be they single-column or multi-column systems, is described in this work. A rational method to conceive, scale-up and compare processes is proposed and illustrated with different examples. All along this article, the results of the equilibrium theory, i.e. obtained with an infinitely efficient column, are used to normalize the system and propose a frame for a rigorous comparison between the different configurations considered in this work. Then, the impact of the fluid velocity, the column length, the refresh time, the sequence organization and the number of columns on the yield, productivity and fluid requirement is investigated. It is found that the optimal process, in terms of number of columns and residence time, depends in the targeted protein recovery. For instance, when considering a titer of 1g/L, 2 columns connected in series in the loading zone are necessary to reach 90% recovery, 3 columns to reach 95% and 4 columns to obtain 99% recovery.

Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements

The goal of this study was to adapt a batch mAb purification chromatography platform for continuous operation. The experiments and rationale used to convert from batch to continuous operation are described. Experimental data was used to design chromatography methods for continuous operation that would exceed the threshold for critical quality attributes and minimize the consumables required as compared to batch mode of operation. Four unit operations comprising of Protein A capture, viral inactivation, flow-through anion exchange (AEX), and mixed-mode cation exchange chromatography (MMCEX) were integrated across two Cadence BioSMB PD multi-column chromatography systems in order to process a 25L volume of harvested cell culture fluid (HCCF) in less than 12h. Transfer from batch to continuous resulted in an increase in productivity of the Protein A step from 13 to 50g/L/h and of the MMCEX step from 10 to 60g/L/h with no impact on the purification process performance in term of contaminant removal (4.5 log reduction of host cell proteins, 50% reduction in soluble product aggregates) and overall chromatography process yield of recovery (75%). The increase in productivity, combined with continuous operation, reduced the resin volume required for Protein A and MMCEX chromatography by more than 95% compared to batch. The volume of AEX membrane required for flow through operation was reduced by 74%. Moreover, the continuous process required 44% less buffer than an equivalent batch process. This significant reduction in consumables enables cost-effective, disposable, single-use manufacturing.

Sample displacement chromatography of plasmid DNA isoforms

Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future.

Stability of multi-column systems with initial imperfections and non-linear connections

The stability and second-order response of multi-column system with initial geometric imperfections (i.e., made of columns with initial curvature, out-of-plumb in the plane of bending under eccentric axial loads at both ends), and non-linear end connections subjected to a lateral load at the top floor level are presented in a classical manner. The proposed method is based on the Euler–Bernoulli theory and limited to 2D multi-column systems with sidesway uninhibited or partially inhibited by lateral bracing at the top level. The combined effects of initial imperfections and non-linear connections in the plane of bending are condensed into the proposed governing equations which also can be used to evaluate the induced moments and second-order deflections along each column as the lateral and axial loads are applied to each column at the top level. The non-linear end connections are modeled using the three-parameter model suggested by Richard and Abbott. The bracing force is defined as a function (linear or non-linear) of the lateral sway. Three comprehensive examples are presented in detail that demonstrate the effectiveness of the proposed method and show the combined effects of initial imperfections and non-linear connections on the stability and second-order response of 2D multi-column systems. The proposed method can be used by structural researchers and engineers to investigate these effects on the stability and second-order response of multi-column systems including cases with relatively large lateral sways.

Evaluating the performance of different multicolumn setups for chromatographic separation of proteins on hydrophobic interaction chromatography media by a numerical study

A theoretical study has been performed on the effectiveness of isolating a target component out of a multi-component protein mixture using different arrangements of chromatographic columns. Three continuous systems have been considered which were able to perform solvent gradient separations, such as: open loop simulated moving bed, countercurrent solvent gradient purification and carousel multicolumn setup. The performance of the continuous processes was examined with respect to productivity, yield and eluent consumption and compared to a single-column batch system. As a case study separation of a ternary mixture of proteins on HIC media has been selected. Two separation problems have been analyzed referring to the situation when the target component was the most strongly adsorbed as well as when it exhibited intermediate adsorption strength. A mathematical model has been used to simulate the process dynamics and to optimize operating conditions for the separation. The numerical study indicated that batch column arrangements can outperform SMB-based configurations regarding all performance indicators considered, which has been attributed to solvent mixing in the recycled streams and distortion of the gradient shape in SMB units. It has been concluded that the performance of complex multicolumn systems should be verified vs. batch column operations prior to the realization of the separation process.

Performance limits and kinetic optimization of parallel and serially connected multi-column systems spanning a wide range of efficiencies for liquid chromatography

Using a set of experimentally determined liquid chromatography column performance data, it has been investigated how a range of efficiencies can best be covered when using a multi-column system. Two main variants are considered: a serially-connected variant (realizing different column lengths by connecting a different number of column segments in series) and a parallel-connected variant (realizing different column lengths by simply switching between columns with a different length arranged in parallel). Both variants are compared for their ability to keep the average analysis time along a given range of efficiencies as close as possible to the intrinsic Knox & Saleem-speed limit. It was found that the serial connection mode offers a better compromise between average speed and amount of required silica (total required column length) than the parallel connection mode for all efficiency ranges running from 5000-10,000 plates up to 75,000-150,000 plates. Considering an ultra-high performance liquid chromatography (UHPLC) operation at 1200 bar, the best possible serial connection system can get within about within 15-25% of the Knox & Saleem-speed limit, whereas a three-column parallel system can only get to within 40-50% of the speed limit, while needing 50-100% more total column length. In absolute terms, the serially-connected system with individually optimized segment lengths should be able to cover a range of 5000-75,000 theoretical plates (dynamic range of 25) in an average analysis time of 14.3 min when using a 1200 bar instrument. At 400 bar, this would be 37.9 min, showing that the construction of wide-efficiency range systems would be one of the application areas where the advantages of UHPLC-conditions would be most fully realized.

Stability and second-order non-linear analysis of 2D multi-column systems with semirigid connections: Effects of initial imperfections

An analytical method and closed-form equations that evaluate the elastic stability and second-order response of 2D multi-column systems with initial geometric imperfections (i.e., columns with initial curvature or out-of-straightness and out-of-plumbness in the plane of bending) and semirigid connections subjected to eccentric axial loads and to a lateral load at the top floor level are derived in a classical manner. The proposed method is based on the Euler–Bernoulli theory and limited to 2D multi-column systems with sidesway uninhibited or partially inhibited subjected to gravity loads. The combined effects of initial imperfections and semirigid connections in the plane of bending are condensed into the proposed equations, which can also be used to evaluate the induced elastic bending moments and second-order deflections along each column member of a multi-column system as the lateral and axial loads are applied. The effects of torsion, shear and axial deformations along each column and out-of-plane deformations are not included. Three examples are presented in detail that demonstrate the effectiveness of the proposed method and the corresponding closed form equations showing the importance of initial imperfections, semirigid connections and lateral bracing on the stability and second-order behavior of multi-column systems.

High peak capacity separation of peptides through the serial connection of LC shell‐packed columns

High peak capacity was obtained for the separation of a HSA tryptic digest through the serial connection of three LC columns packed with shell material (C18, 15 cmx4.6 mm, 2.7 mum particle size). A major benefit of these particles, consisting of a 1.7 mum solid core and a 0.5 mum porous shell, is the small diffusion path, which reduces axial dispersion of solutes and minimizes peak broadening allowing for higher resolving power to be obtained. Increase in the temperature decreases the viscosity and the backpressure on the columns and in this way allowed to couple multiple columns. Single and multicolumn systems were first evaluated for the separation of a standard sample mixture in the isocratic mode; afterwards three columns could be coupled at 60 degrees C, generating more than 90,000 effective plates (200,000 plates/meter) for the separation of HSA in gradient mode on a conventional HPLC instrument. Peak capacity ratios were found in good accordance with the theoretical gain in resolution, i.e. values of 1.37 and 1.67 were measured, when doubling and tripling the length of stationary phases, respectively (the highest measured peak capacity on three coupled columns was 367).

Tension and Compression Stability and Second-Order Analyses of Three-Dimensional Multicolumn Systems: Effects of Shear Deformations

The stability and second-order analyses of three-dimensional 3D multicolumn systems including the effects of shear defor- mations along the span of each column are presented in a condensed manner. This formulation is an extension to an algorithm presented recently by the writer in 2002 and 2003 by which the critical load of each column, the total critical load, and the second-order response of a 3D multicolumn system with semirigid connections can be determined directly. The proposed solution includes not only the combined effects of flexural deformations and shear distortions along the columns in their two principal transverse axes, but also the effect of the shear forces along each member induced by the applied end axial force as the columns deform and deflect as suggested by Haringx in 1947 and explained by Timoshenko and Gere in 1961 in their two principal transverse axes. The extended characteristic transcendental equations corresponding to multicolumn systems with sidesway and twist uninhibited, partially inhibited, and totally inhibited that are derived and discussed in this publication find great applications in the stability and second-order analyses of 3D multicolumn systems made of materials with relatively low shear stiffness such as orthotropic composite materials fiber reinforced plastic and multilayer elastomeric bearings used for seismic isolation of buildings. The phenomenon of buckling under axial tension in members with relatively low shear stiffness observed by Kelly in 2003 in multilayer elastomeric bearings, and recently discussed by the writer in 2005 is captured by the proposed method. Tension buckling must not be ignored in the stability analysis of multicolumn systems made of columns in which the shear stiffness GAs is of the same order of magnitude as 2 EI/h 2 .

Arrayed miniature electron beam columns for mask making

A significant problem facing integrated circuit mass-production lithography at future technology generations is the throughput of mask writers. Novelx has developed the technologies to build a distributed multiaxial parallel beam system consisting of a monolithic array of individually correctable electron beam columns. These technologies can be scaled to manufacture a system with a sufficient number of columns to write a single mask at the 32nm node with a write time of an order of magnitude faster than what is forecasted at the back end of the 45nm node. The key to these technologies is a new type of miniature electron beam column fabricated and assembled using manufacturable processes. Both single and 1×4 multicolumn systems have been assembled and tested, and have demonstrated 90nm lithography. The columns in these systems operate at low voltage and are all electrostatic. The lenses are monolithically fabricated bonded stacks of micromachined silicon and glass. The monolithic lenses are assembled onto a single package. The multicolumn lens stacks consist of four independent and electrically isolated lenses. The silicon and package are scalable for continued miniaturization, and can be fabricated in larger monolithic arrays of individually correctable beams. The authors show that the 1×4 column system currently in operation can be scaled to a 4×4 column system and that a mask writer using the 4×4 column system is capable of writing a single mask at the 32nm dynamic random access memory 1∕2 pitch node in 10h.