Influence of column design on process-scale ion-exchange chromatography

The performance of two new designs of pump-packed axial flow process chromatography columns have been evaluated for the preparative anion-exchange chromatography of hen egg-white proteins using Whatman Express-Ion Exchanger Q. A 16 1 Side-Pack column and a 24 1 IsoPak column containing Express-Ion Q were used in this study. In each case ca. 20 1 feedstock containing 5-7 g protein/l, was applied per litre packed bed at flow-rates of ca. 150 and 300 cm/h. In each case the ovalbumin binding capacity was ca. 70 g/l packed bed with ca. 100% (w/w) recovery of applied protein. A clean-in-place procedure involving storage in 0.5 M NaOH was effective in maintaining chromatographic performance in all cases. These data were consistent with our previous work using the more traditionally configured slurry-packed axial flow columns. Each of these column designs were easy to use facilitating rapid packing with this adsorbent and in the case of IsoPak rapid pump unpacking. The introduction of these column designs significantly improves the task of column packing, hitherto a labour intensive, physically demanding and potentially unreproducible process.     

New Q membrane scale-down model for process-scale antibody purification

Process-scale antibody production requires polishing steps with extremely high product throughput and robust operation. In this communication, the Sartobind Q membrane adsorber for process-scale antibody production is evaluated as an alternative to Q column chromatography. Although the capacity seen with large-scale membrane adsorbers is competitive with column chromatography, the same throughput is not achieved with the current scale-down models. The operational issues currently found in membrane scale-down models, including backpressure, which significantly compromises the membrane's capacity, were examined. A new scale-down model was designed to mimic the liquid flow path found in the large-scale capsule, and a new process capacity equivalent at both small and large scale was successfully achieved. Results of a 4-model virus study with a redesigned Sartobind Q absorber scale-down model at the new process capacity are presented.     

Model-based optimization of a preparative ion-exchange step for antibody purification

A method using a model-based approach to design and optimize an ion-exchange step in a protein purification process is proposed for the separation of IgG from a mixture containing IgG, BSA and myoglobin. The method consists of three steps. In the first step, the model is calibrated against carefully designed experiments. The chromatographic model describes the convective and dispersive flow in the column, the diffusion in the adsorbent particles, and the protein adsorption using Langmuir kinetics with mobile phase modulators (MPM). In the second step, the model is validated against a validation experiment and analyzed. In the third and final step, the operating conditions are optimized. In the optimization step, the loading volume and the elution gradient are optimized with regard to the most important costs: the fixed costs and the feed cost. The optimization is achieved by maximizing the objective functions productivity (i.e. the production rate for a given amount of stationary phase) and product yield (i.e. the fraction of IgG recovered in the product stream). All optimization is conducted under the constraint of 99% purity of the IgG. The model calibration and the analysis show that this purification step is determined mainly by the kinetics, although as large a protein as IgG is used in the study. The two different optima resulting from this study are a productivity of 2.7 g IgG/(s m3) stationary phase and a yield of 90%. This model-based approach also gives information of the robustness of the chosen operating conditions. It is shown that the bead diameter could only be increased from 15 microm to 35 microm with maximum productivity and a 99% purity constraint due to increased diffusion hindrance in larger beads.     

Constrained optimization of a preparative ion-exchange step for antibody purification

Today, the optimization of chromatographic separation is usually based on experimental work and rule of thumb. The process and analytical technology (PAT) initiative, of the US Food and Drug Administration, has provided the opportunity of using model-based approach when designing downstream processing of pharmaceutical substances. A nonlinear chromatography model was used in this study to optimize a preparative ion-exchange separation step involving two components. Separation was simulated with the general rate model employing Langmuir kinetics. Optimization was performed with an indirect method allowing constraints on the purity, thus avoiding sub-optimization, which can lead to noisy objective functions. The six decision variables used in the optimizations were flow rate, loading volume, initial salt concentration in the elution, final salt concentration in the linear elution gradient and the two cut points. A graphical representation of the effect of the decision variables on the objective function was used to verify that the optimization had converged to the true optimum. The optimal operating points, using productivity and yield separately as objective functions, were found and compared with the product of productivity and yield as objective function. The optimum obtained with this objective function had a lower productivity, than the productivity function, but much higher yield, which makes it a good substitute for a cost function.     

New 18-l process-scale counter-current chromatography centrifuge

A new Dynamic Extractions Maxi-counter-current chromatography (CCC) centrifuge with a column volume of 18-l has been installed in the Advanced Bioprocessing Centre at Brunel. This instrument has four times the capacity of the 4.6-l Maxi-CCC centrifuge which has been operating robustly for 3 years. Tests using the model sample system benzyl alcohol and p-cresol with a heptane:ethyl acetate:methanol:water (HEMWat) phase system (1.4:0.1:0.5:1.0) show that resolution is almost double with this new high capacity device. Commissioning tests with a mixture of caffeine, K_D = 0.21; ferulic acid, K_D = 0.82; umbelliferone, K_D = 1.2 and vanillin, K_D = 1.49 using a HEMWat phase system of 1:1.5:1:1.5 on the 9-l column show that resolutions equivalent to analytical instruments will be possible using the full 18-l capacity. They also show that predictable scale-up from simple test tube tests is feasible with knowledge of the stationary phase retention for the planned process scale run.     

Cored anion-exchange chromatography media for antibody flow-through purification

Agarose-based anion-exchangers (e.g. quaternary amine, Q) have been widely used in monoclonal antibody flow-through purification to remove trace levels of impurities. Such media are often packed in a large column and the operation is usually robust but with limited throughput due to the compressibility of agarose and consequentially low bed permeability. In order to address this limitation, cored Q beads consisting of a rigid core and a thin agarose gel coating were developed and evaluated for protein flow-through chromatography. Using laboratory-scale columns it was found that, the cored beads indeed provide significantly enhanced rigidity and flow permeability relative to conventional homogeneous agarose resins. Depending on the structure and size of the cored beads, the permeability was 2-4-fold higher than that of a commonly used commercial agarose resin. Good virus and host cell protein clearance was achieved with the cored Q beads even at increased flow velocities. In addition, the impermeable core allows for more efficient use of buffers without loss of useful capacity in polishing applications. Process analyses based upon the experimental data demonstrated that the enhanced permeability achieved with the cored beads can significantly improve process throughput and economics.     

Rapid monoclonal antibody adsorption on dextran-grafted agarose media for ion-exchange chromatography

The binding capacity and adsorption kinetics of a monoclonal antibody (mAb) are measured for experimental cation exchangers obtained by grafting dextran polymers to agarose beads and compared with measurements for two commercial agarose-based cation exchangers with and without dextran grafts. Introduction of charged dextran polymers results in enhanced adsorption kinetics despite a dramatic reduction of the accessible pore size as determined by inverse size-exclusion chromatography. Incorporation of neutral dextran polymers in a charged agarose bead results instead in substantially lower binding capacities. The effective pore diffusivities obtained from batch uptake curves increase substantially as the protein concentration is reduced for the resins containing charged dextran grafts, but are much less dependent on protein concentration for the resins with no dextran or uncharged dextran grafts. The batch uptake results are corroborated by microscopic observations of transient adsorption in individual particles. In all cases studied, the adsorption kinetics is characterized by a sharp adsorption front consistent with a shell-progressive, diffusion limited mechanism. Greatly enhanced transport rates are obtained with an experimental resin containing charged dextran grafts with effective pore diffusivities that are 1-9 times larger than the free solution diffusivity and adsorption capacity approaching 300 mg/cm3 of particle volume.     

Membrane affinity chromatography for analysis and purification of biopolymers

Summary Affinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only about 2.5 min for analysis at a flow-rate of 1.0 mL min⁻¹. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 mL min⁻¹ within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid. It can also be used for micro-scale purification of biopolymers.     

Improvement purification of sulfated oligofucan by ion-exchange displacement centrifugal partition chromatography

Centrifugal partition chromatography in ion-exchange displacement mode was used to fractionate mixtures of sulfated oligofucans obtained by partial depolymerization of brown seaweed fucoidans. Diluted (10%, v/v) protonated LA2 (a lipophilic secondary amine) is used as a weak exchanger. In an attempt to improve this method, several solvents (methyl isobutyl ketone, methyl tert.-butyl ether, BuOH) were tested to dissolve LA2H+. MtBE produced less bleeding than MiBK, whereas BuOH proved unsuitable. The sample injected needs to be highly diluted in water to ensure participation in the chromatographic process. A comparison of data (NMR, composition, molecular mass) indicated the homogeneity of the fractions obtained as well as the differences between them.     

Optimization of monoclonal antibody purification by ion-exchange chromatography. Application of simple methods with linear gradient elution experimental data

Simple methods for the optimization of ion-exchange chromatography of proteins in our previous papers were applied to cation-exchange chromatography purification of monoclonal antibodies (Mab). We carried out linear gradient elution experiments, and obtained the data for the peak salt concentration and the peak width. From these data, the distribution coefficient as a function of salt concentration, and the height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity were calculated. The optimized linear gradient elution conditions were determined based on the relationship between buffer consumption and separation time. The optimal stepwise elution conditions were determined based on the relationship between the distribution coefficient and the salt concentration.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

Multi-dimensional chromatography using on-line coupled microcolumn liquid chromatography—capillary gas chromatography for quantitative pesticide residue analysis

Highly selective separations can be achieved by utilizing different separation modes in a multi-dimensional (on-line coupled) chromatographic system. The application of such a system utilizing microcolumn liquid chromatography coupled on-line to capillary gas chromatography for the determination of 2-(1,1-dimethylethyl)-5-pyrimidinol, a pesticide metabolite, in various corn matrices at the ng g^?1 level is described. A comparative quantitative study with conventional manual sample pretreatments followed by capillary gas chromatography-mass spectrometry indicated equivalence between the two techniques. The system offers the advantages of reduced sample handling steps and analysis time, high potential for automation and adequate sensitivity.     

Strong ion-exchange centrifugal partition chromatography as an efficient method for the large-scale purification of glucosinolates

The glucosinolates sinalbin and glucoraphanin were purified by strong ion-exchange displacement centrifugal partition chromatography (SIXCPC). The optimized conditions involved the biphasic solvent system ethyl acetate/n-butanol/water (3:2:5, v/v), the lipophilic anion-exchanger Aliquat 336 (trioctylmethylammonium chloride, 160 and 408 mM) and a sodium iodide solution (80 and 272 mM) as displacer. Amounts as high as 2.4 g of sinalbin and 2.6g of glucoraphanin were obtained in one step in 2.5 and 3.5h respectively, starting from 12 and 25 g of mustard and broccoli seed aqueous extracts, using a laboratory scale CPC column (200 mL inner volume).     

Evaluating and monitoring the packing behavior of process-scale chromatography columns

The packing characteristics of process-scale chromatography columns were evaluated using the responses to conductivity-based pulse and step inputs derived from tracer experiments and in-process transitions (i.e. column equilibration and regeneration steps). Characteristics of the measured residence time distributions (RTDs) were quantified by statistical moments and using the equations derived from the Gaussian model. The first and second moments calculated from in-process step transitions for multiple runs were in good agreement with those moments calculated from the pulse-input experiments conducted immediately after column packing. This indicates that most of the time the bed behavior at the time of packing is consistent with that at the time of operation. Due to the significant resistance to protein mass transfer inside the particles, estimated plate heights for protein solutes are expected to be much greater than those observed from the experiments using saltbased tracers. Thus, the column efficiency derived from salt-based experiments can be a useful measure of packing consistency rather than a significant parameter influencing the outcome of protein separations.     

Protein-surface interaction maps for ion-exchange chromatography

In this paper, protein-surface interaction maps were generated by performing coarse-grained protein-surface calculations. This approach allowed for the rapid determination of the protein-surface interaction energies at a range of orientations and distances. Interaction maps of lysozyme indicated that there was a contiguous series of orientations corresponding to several adjacent preferred binding regions on the protein surface. Examination of these orientations provided insight into the residues involved in surface interactions, which qualitatively agreed with the retention data for single-site mutants. Interaction maps of lysozyme single-site mutants were also generated and provided significant insight into why these variants exhibited significant differences in their chromatographic behavior. This approach was also employed to study the binding behavior of CspB and related mutants. The results indicated that, in addition to describing general trends in the data, these maps provided significant insight into retention data of the single-site mutants. In particular, subtle retention trends observed with the K12 and K13 mutants were well-described using this interaction map approach. Finally, the number of interaction points with energies stronger than -2 kcal/mol was shown to be able to semi-quantitatively predict the behavior of most of the mutants. This rapid approach for calculating protein-surface interaction maps is expected to facilitate future method development for separating closely related protein variants in ion-exchange systems.     

Rational design of a new one-step purification strategy for Candida antarctica lipase B by ion-exchange chromatography

A fast and efficient one-step method for purification of lipase B from Candida antarctica by ion-exchange chromatography was developed by rational design. The electrostatic properties of the enzyme were calculated and validated by isoelectric focusing and measurement of the titration curve. C. antarctica lipase B shows an unusual pH profile with a broad isoelectric region from pH 4 to 8. At pH 3 C. antarctica lipase B can be bound to a cation-exchange chromatography column and was purified to homogeneity with a purification factor of 2.4. It was stable at pH 3, the residual activity was still 80% after 6 days incubation at 20 degrees C. The broad isoelectric region of C. antarctica lipase B is unique as compared to almost all other alpha/beta-hydrolases which have a well-defined isoelectric point. A search in the lipase engineering database resulted in only one further alpha/beta-hydrolase, the Fusarium solani cutinase, which also has a broad isoelectric region.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Thin-layer chromatography in food and agricultural analysis

The TLC analysis of agricultural products, foods, beverages, and plant constituents is reviewed for the period from mid-1995 to mid-1999. Techniques and applications for a wide range of analyte and sample matrix types are covered, with specification of the particular layers, mobile phases, detection methods, and quantification conditions in many cases.