Continuous bind‐and‐elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation

Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938–948, 2016     

A framework for the prediction of scale-up when using compressible chromatographic packings

Experimental data are given for the solid pressure distributions in chromatography columns of various column aspect ratios packed with four types of agarose-based resin. The loss of column wall support at large scales can result in unexpectedly high pressures caused by the compression of the matrix via drag forces exerted by fluid flow through the bed. The need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation. Several studies have generated correlations that allow for the prediction of column pressure drops, but they either are mathematically complex, which impairs their practical use, or require a large number of experiments to be performed before they can be used. In this study an empirical correlation was developed based on a previously proposed model, which links the critical velocity of operation of a chromatographic system (microcrit), to the gravity-settled bed height (L0), the column diameter (D), the feed viscosity (micro), and the compressibility of the chromatographic media used (micro 10%). The methodology developed in this study is straightforward to use and significantly reduces the burden of preceding laboratory-scale experimentation. The approach can be used to predict the critical velocity of any chromatographic system and will be useful in the development of chromatographic operations and for column sizing.     

Examination of protein adsorption in fluidized bed and packed bed columns at different temperatures using frontal chromatographic methods

The influences of various experimental parameters on the dynamic adsorption capacity (DAC) and the dynamic adsorption rate (DAR) of a biomimetic affinity silica-based adsorbent in fluidized and packed bed columns operated under plug flow conditions and at different temperatures have been investigated with different inlet concentrations of hen egg white lysozyme (HEWL) and human serum albumin (HSA). The DACs as well as the DARs of both the fluidized and packed beds were examined at 10% saturation (i.e., at the QB value) and the experimental data compared with the corresponding data obtained from batch equilibrium adsorption procedures. Parameters examined included the fluid superficial velocity and protein concentration and their effect on the binding capacity and column efficiency. Consistent with various results reported from this and other laboratories on the behavior of biospecific affinity adsorbents derived from porous silica and zirconia particles, adsorbents prepared from Fractosil 1000 were found to exhibit appropriate rheological characteristics in fluidized bed systems under the experimental conditions. Moreover, changes in temperature resulted in a more significant effect on the breakthrough profiles of HSA compared to HEWL with the immobilized Cibacron Blue F3G-A with Fractosil 1000 adsorbent. This result suggests that temperature effects can possibly be employed profitably in some processes as part of a strategy to enhance column performance with fluidized bed systems for selective recovery of target proteins. At relatively low superficial velocities of the feed, the DARs with HEWL and HSA were similar for both the fluidized and packed bed column systems, whereas, at high superficial velocities, the DARs for these proteins were larger with the packed bed columns.     

Microbial production of 2-chloro-alpha-methylbenzyl alcohol and its adsorptive recovery

An adsorptive process was combined with yeast-mediated production of chiral 2-chloro-alpha-methylbenzyl alcohol (o-Cl-1-PA) for effective product recovery and reuse of the reaction medium. Low temperature was suitable for long-term reactor operation, and continuous production using a shallow-bed reactor was achieved for at least 22 days while maintaining a high conversion. The appropriate size of the adsorption column for product recovery from the reactor effluent was estimated through measurement of breakthrough curves of o-Cl-1-PA in a packed bed of the resin at various adsorbate concentrations and feed flow rates. Using the adsorption column, 98% of the product and the residual substrate were recovered from the reactor effluent, and the effluent from the adsorption column was successfully reused as the reaction medium after microfiltration to save the medium consumption.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

New system for preparative electrochromatography of proteins

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.     

Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange

Extraction and purification of high‐value minor proteins directly from milk without pre‐treatment is a challenge for the dairy industry. Pre‐treatment of milk before extraction of proteins by conventional packed‐bed chromatography is usually necessary to prevent column blockage but it requires several steps that result in significant loss of yield and activity for many minor proteins. In this paper, we demonstrate that it is possible to pass 40–50 column volumes of various milk samples (raw whole milk, homogenized milk, skim milk and acid whey) through a 5 mL cryogel chromatographic column at 550 cm/h without exceeding its pressure limits if the processing temperature is maintained above 35°C. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg/mL) was similar to that of the binding capacity (2.01 mg/mL) at equilibrium with 0.1 mg/mL of lactoferrin in the feed samples. The cryogel column selectively binds lactoferrin and lactoperoxidase with only minor leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of over 85% and a purity of more than 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed‐bed chromatography for direct capture of proteins from milk. Biotechnol. Bioeng. 2009;103: 1155–1163. © 2009 Wiley Periodicals, Inc.     

Restricted diffusion effect on the binding of proteins to porous polymer resins as studied by repetitive injection method

A solution of bovine serum albumin (BSA) is repeatedly injected into a column packed with highly porous and hydrophobic polymer resins at appropriate intervals. The injected BSA is thoroughly retained in the column for 10 injections and, afterwards, starts to be eluted from the column gradually. Taking into consideration the restricting effect of already bound BSA upon the diffusion of newly injected BSA into the pores of the polymer resins, we can interpret the BSA elution profile from columns packed with polymer resin of various pore sizes and porosities. The effects of the binding rate constant and BSA concentration upon the elution profiles of BSA are also analyzed. Formyl groups are introduced into the polymers as a binding site with proteins, and the elution profiles of BSA from the column packed with the formylated resin are also analyzed.     

High-performance liquid chromatography using novel square tile-shaped hydroxyapatite crystals as adsorbent

High-performance liquid chromatography using, as adsorbent, novel square tile-shaped hydroxyapatite crystals (with thicknesses of about 2 microns and diameters of 3-7 microns) has been developed. The chromatographic efficiencies of the novel hydroxyapatite packed columns are almost equal to those of the previously developed spherical hydroxyapatite packed columns; high chromatographic resolutions can be obtained by using extremely reduced column lengths of 0.5-3 cm. Since both the square and the spherical hydroxyapatite have roughly the same particle size of some micrometers, the chromatographic efficiency can be deduced to be determined mainly by the particle size rather than the particle shape.     

Evaluating two process scale chromatography column header designs using CFD

Chromatography is an indispensable unit operation in the downstream processing of biomolecules. Scaling of chromatographic operations typically involves a significant increase in the column diameter. At this scale, the flow distribution within a packed bed could be severely affected by the distributor design in process scale columns. Different vendors offer process scale columns with varying design features. The effect of these design features on the flow distribution in packed beds and the resultant effect on column efficiency and cleanability needs to be properly understood in order to prevent unpleasant surprises on scale‐up. Computational Fluid Dynamics (CFD) provides a cost‐effective means to explore the effect of various distributor designs on process scale performance. In this work, we present a CFD tool that was developed and validated against experimental dye traces and tracer injections. Subsequently, the tool was employed to compare and contrast two commercially available header designs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:837–844, 2014     

Preparative liquid chromatography of carbohydrates: mono- and di-saccharides, uronic acids, and related derivatives

The general principles and practical aspects of preparative high-performance liquid chromatography (l.c.) of mono- and di-saccharides, sugars acids, lactones, and N-acetylated amino sugar derivatives are described. Milligram to gram quantities of these carbohydrates were isolated on semi-preparative (0.78 X 30 cm) or preparative (approximately 2.0 X 30 cm) columns packed with aminopropyl silica gel provided better resolution of individual mono- and di-saccharides, but columns of cation-exchange resin had higher capacity and were more durable and economical to use. Preparative, cation-exchange columns were operated at flow rates of less than 5 mL/min and pressures of approximately 1-2 MPa, allowing them to be used on unmodified analytical l.c. systems. Details are given for the efficient packing, use, and care of these columns, and on the effects of column selectivity, packing technique, and sample size on chromatographic resolution. Isolation of naturally occurring sugars from biological sources on a laboratory-packed column is described.     

Chromatography of human prothrombin from Nitschmann fraction III on Q Sepharose Fast Flow using axial and radial flow column

An axial column (Hitrap Q 5 ml, 2.5 2 1.6 cm) and a radial flow column (3.5 2 5 cm) packed with Q Sepharose Fast Flow media had been evaluated for the separation of human prothrombin. Nitschmann fraction III dissolved in buffered saline (0.10 M sodium chloride buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Effects of sample flow rate of the two columns were screened. Under radial flow conditions using the radial column, sample flow rate up to 15 ml/min (i.e. 18 bed volumes/h) was achieved and the operating pressure was below 0.2 MPa eventhough the elution velocity was 30 ml/min. Breakthrough capacity was determined by analyzing the total protein and prothrombin activity of the target protein-containing fraction under subsaturating conditions and both columns had almost the same breakthrough capacity per ml media, indicating that the sample loading was independent of radial column geometry. It was concluded that the radial column is an attractive alternate to traditional axial packed bed column, exhibiting very good potential for use in the separation of human prothrombin.     

Clarification and capture of high‐concentration refold pools for E. coli‐based therapeutics using expanded bed adsorption chromatography

Expanded bed adsorption (EBA) chromatography was investigated for clarification and capture of high‐concentration refold pools of Escherichia coli‐based therapeutics. Refolding of denatured inclusion bodies (IBs) at high protein concentration significantly improved product throughput; however, direct filtration of the refold materials became very challenging because of high content of protein precipitates formed during refolding. In addition, irreversible protein precipitation caused by high local concentration was encountered in packed bed capture during cation exchange chromatography elution, which limited column loading capacity and capture step productivity. In this study, the two issues are addressed in one unit operation by using EBA. Specifically, EBA can handle feed streams with significant amount of particles and precipitates, which eliminated the need for refold pool clarification through filtration. The relatively broad EBA elution profile is particularly suitable for proteins of low solubility and can effectively avoid product loss previously associated with on‐column precipitation during capture. As the EBA resin (RHOBUST^® FastLine SP IEX) used here has unique properties, it can be operated at high linear velocity (800–1,600 cm/h), while achieving a selectivity and impurity clearance largely comparable to the packed bed resin of the same ligand chemistry (SP Sepharose FF). Furthermore, the filtration of the EBA elution pool is easily manageable within facility capability. Overall, this study demonstrates that the EBA process helps debottleneck the purification of high‐turbidity refold pools by removing precipitates and concurrently capturing the product, which can be applied to other E. coli‐based therapeutics that also requires refolding of IBs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:113–123, 2014     

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.     

Endostatin capture from Pichia pastoris culture in a fluidized bed. From on-chip process optimization to application

One of the characteristics of the methylothrophic yeast Pichia pastoris is its ability to grow to a very high cell density. Biomass concentrations of 300-400 g wet mass/l are common. It is therefore obvious that the recovery processes of extracellular proteins from this microorganism should take into account the effect of high biomass content. Separation by filtration and/or centrifugation is possible but these steps are cumbersome and can affect the protein recovery. The use of fluidized beds is attractive proteins capture option since it eliminates the biomass while capturing the desired protein. Zirconia-based resins possess unique properties which make them appropriate for processing high biomass concentrations in an expanded bed mode. The beads are particularly heavy (density is 3.2 g/ml) and small (75 microm) and therefore can accommodate high fluidization velocity and high mass transport. Specific operating conditions for effective capture of expressed protein have to be determined. This determination is generally time consuming and requires relatively large amount of feedstock for the lab trials. To avoid multiple chromatographic trials in columns, optimal conditions of adsorption and elution were determined by ProteinChip technology coupled with mass spectrometry. This technology involves flat chip surfaces functionalized as chromatographic beads where it is possible to adsorb and desorb proteins. Four different functional groups (strong anion-exchange, weak cation-exchange, hydrophobic and metal chelate) were tested and the retained proteins were analyzed directly by mass spectrometry. The weak cation-exchange group was chosen for further work. The Zirconia-based weak cation-exchange sorbent (CM HyperZ) was evaluated for binding capacity in a packed column and then for capturing endostatin from crude feed stock. Based on the previously determined conditions; 45 l of culture containing approximately 15 kg of biomass (wet mass) and 3 g endostatin were applied on an expanded bed at a flow-rate of 535 cm/h, yielding 80% of the endostatin and removing approximately 80% of foreign proteins.     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand

Due to the peculiar composition of the egg yolk and the lack of specific affinity ligands, Y immunoglobulins are normally purified using complex and time consuming procedures involving a combination of precipitation and chromatographic steps first to extract and capture and then to polish IgY. In this study, we have examined the applicability for IgY affinity purification of TG19318, a synthetic ligand for immunoglobulin, obtained from the screening of combinatorial libraries, and already characterized for its capability to purify immunoglobulins of class G, M, E and A. Soluble proteins were separated from the lipidic fraction of egg yolk by the water dilution method and loaded on to TG19318 affinity columns prepared by immobilizing the ligand on the commercially available support Emphaze™. In a single chromatographic step TG19318 affinity columns led to an efficient capture of IgY directly from crude samples, and with a purity degree higher than 90%, as determined by densitometric scanning of SDS–PAGE analysis of bound fractions, and with full recovery of antibody activity, as determined by ELISA assay. Higher recovery and purity of IgY was obtained by using loading buffers at pH close to 6.5. Column capacity, determined by applying 4× excess IgY to 1 ml bed volume column, and eluting the retained immunoglobulins, was close to 65 mg of IgY per ml of resin. Chemical and chromatographic stability of TG19318/Emphaze was tested before and after various treatments. The derivatized matrix was found to be very stable, in terms of ligand leakage and maintenance of IgY binding capacity, under conditions of normal column usage, cleaning and storage.     

High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.     

Sorbitol determination by liquid chromatography: application to red blood cells of diabetic rats

This report describes an application of liquid chromatography to the determination of sorbitol in red blood cells. The chromatograph employed in the present study was made up of sub- and main-separation systems and a detector portion. The sub-separation system was for concentration of polyols and involved two small columns, each containing the same anion exchange resin. The first was a tiny column which, in borate form, served as the concentrator of polyols and sugars charged in a large volume, while the second, in acetate form, separated the carbohydrates from the borate. The main system was for the fine separation of each carbohydrate and employed cation exchange columns. The detector part utilized a flow fluorometric method comprising two successive reactions: periodate oxidation followed by the Hantzsch reaction. The resulting whole chromatographic system was applied to the determination of sorbitol in red blood cells obtained from normal rats and rats made diabetic by the administration of streptozotocin; a part of the latter group had also received an aldose reductase inhibitor. Our results supported the concepts that a prolonged duration of high blood glucose level induces an elevated level of sorbitol inside red blood cells and that aldose reductase inhibitors are effective in reducing this level.