Dynamic Binding Capacity Values Research Articles

Moving adsorption belt system for continuous bioproduct recovery utilizing composite fibrous adsorbents.

A continuous protein recovery and purification system based on the true moving bed concept is presented. A novel adsorbent material, in the form of an elastic and robust woven fabric, served as a moving belt following the general designs observed in known belt conveyors. The composite fibrous material that forms the said woven fabric showed high protein binding capacity, reaching a static binding capacity equal to 107.3mg/g, as determined via isotherm experiments. Moreover, testing the same cation exchange fibrous material in a packed bed format resulted in excellent dynamic binding capacity values (54.5mg/g) even when operating at high flow rates (480cm/h). In a subsequent step, a benchtop prototype was designed, constructed, and tested. Results indicated that the moving belt system could recover a model protein (hen egg white lysozyme) with a productivity up to 0.5mg/cm2/h. Likewise, a monoclonal antibody was directly recovered from unclarified CHO_K1 cell line culture with high purity, as judged by SDS-PAGE, high purification factor (5.8), and in a single step, confirming the suitability and selectivity of the purification procedure.

Dynamic adsorption/desorption of proteins with thermo-responsive polymer grafted sepharose fast flow sorbents

This investigation has focused on the dynamic adsorption and desorption behaviour of proteins with thermo-responsive co-polymer grafted chromatographic materials. To that end, the dynamic adsorption performance of bovine holo-lactoferrin (bhLf) with two thermo-responsive chromatographic sorbents, i.e. poly (N-isopropyl acrylamide-co-tert-butylacryl amide-co-acrylic acid) grafted Sepharose Fast Flow (ITA) and poly (NIPAAm-co-tert-butylacryl amide-co-2-(diethylamino)ethyl methacrylate)) grafted Sepharose Fast Flow (ITD) at two different column temperatures and different protein loading conditions was examined by frontal chromatographic analysis methods. The dynamic binding capacity (DBC), dynamic binding rate (DBR) and column efficiency of the ITA sorbent for bhLf at 50 °C were greater than those at 20 °C. The dynamic binding characteristics were observed to be influenced by the protein feed concentrations, showing an increase of binding capacity with an increase in the bhLf feed concentration. A large proportion (≥65%) of the bhLf molecules adsorbed onto the ITA sorbent could be eluted from the column by simply lowering the column temperature from 50 °C to 20 °C. The corresponding DBC values of ITA sorbent for bovine serum albumin (BSA) were much lower than those for bhLF at both 20 °C and 50 °C due to electrostatic repulsion effects between the negatively charged BSA and the negatively charged co-polymer modified surface of the ITA sorbent, whilst the opposite trend was found with positively charged co-polymer (i.e. poly (N-isopropyl acrylamide-co-tert-butylacryl amide-co-2-(diethyl-amino)ethyl methacrylate)) grafted Sepharose Fast Flow (ITD), indicative of different separation selectivity.

Implications from the grafting density and ionic capacity effects on protein adsorption to poly (N,N-dimethylaminopropyl acrylamide)-grafted sepharose FF

Our previous study reported a novel protein ion-exchanger, N,N-dimethylaminopropyl acrylamide (DMAPAA)-grafted Sepharose FF, of very high protein adsorption capacity and uptake rate. To gain new insight into the polymer grafting effects on protein adsorption and chromatography, 18 DMAPAA-grafted anion exchangers of four grafting densities and various chain lengths (i.e., ionic capacities, ICs) were prepared and named as FF-Brx-pDMAPAAn (x represents grafting density and n stands for IC in mmol/L). It was found that an optimal IC existed at each grafting density to present a maximal capacity. Of the four optimal-IC resins, FF-Br3-pDMAPAA587 showed the highest capacity (377 mg/mL), but the protein uptake rate, defined as the ratio of effective pore diffusivity to free diffusivity (De/D0), was smaller than the resin of a similar ionic capacity but lower grafting density (FF-Br2-pDMAPAA592). Consequently, the dynamic binding capacity (DBC) of FF-Br2-pDMAPAA592 was higher than that of FF-Br3-pDMAPAA587 at flow velocities higher than 450 cm/h. Moreover, FF-Br3-pDMAPAA587 showed higher adsorption capacity than FF-Br2-pDMAPAA592 at 0−150 mmol/L NaCl, leading to its higher DBC values as compared to FF-Br2-pDMAPAA592 in the same salt concentration range. Taken together, DMAPAA-grafted resins of different grafting densities would be useful for purifying feedstocks of different ionic strengths.

Sequential alginate grafting and sulfonation significantly improve the performance of alginate-grafted Sepharose FF for protein chromatography

Alginate (Alg)-grafted Sepharose FF with an ionic capacity (IC) of 230 mmol/L (Alg-FF-230) was first prepared and then sulfonated to synthesize two cation exchangers of higher ICs (270 and 370 mmol/L, denoted as S-Alg-FF-IC). It was found that lysozyme adsorption capacity increased with increasing IC value, but the uptake rate (De/D0) on S-Alg-FF-370 was small (0.070) at 0 mmol/L NaCl because of the strong binding that limited surface diffusion of the bound protein. However, the dynamic binding capacity (DBC) of S-Alg-FF-370 was much higher in the salt concentration range of 0–150 mmol/L than the lower IC resins due to its high adsorption capacity and increased uptake rate. Especially at 150 mmol/L NaCl, the DBC of S-Alg-FF-370 was about 7.3 times higher than Alg-FF-230 and kept over 70 mg/mL at the flow rate up to 1350 cm/h. The results proved the high salt-tolerant feature of S-Alg-FF-370. By contrast, S-Alg-FF-270 showed higher DBC values than S-Alg-FF-370 in the same flow rate range at 50 mmol/L NaCl. Hence, S-Alg-FF-270 and S-Alg-FF-370 displayed different properties that would make them favorable for use in protein purification at different salt concentrations. The work thus proved that the sequential alginate grafting and sulfonation strategy is promising for creation of ion exchangers for high-performance and cost-effective purification of proteins from feedstocks of different ionic strengths.

Implications from protein adsorption onto anion- and cation-exchangers derivatized by modification of poly(ethylenimine)-Sepharose FF with succinic anhydride

Poly(ethylenimine) (PEI)-grafted Sepharose FF with an ionic capacity (IC) of 740 mmol/L (FF-PEI-L740) was modified with succinic anhydride, and six mix-charged resins of different ionic capacities (ICs) were synthesized [denoted as “Am-Cn” according to their anion exchange groups (amino groups) and cation exchange groups (carboxyl groups)]. Protein adsorption behaviors were investigated using bovine serum albumin (BSA) and lysozyme (LZM) as model proteins. The modification led to the net positive charge decrease to approximately zero (A320-C270), and then reversed the net charge to negative till the maximum negatively charge resin A20-C970. Adsorption capacity for BSA decreased from 205 ± 5 mg/mL (FF-PEI-L740) to 65 ± 3 mg/mL (A320-C270), but increased, for LZM, from 31 ± 1 mg/mL (A320-C270) to 281 ± 13 mg/mL (A20-C970) with increasing net negative charge. The uptake rate of BSA or LZM decreased with decreasing net positive charge or increasing net negative charge due to the decreased flexibility of the PEI chains brought from the increased electrostatic attraction or repulsion and/or tight binding due to the high net charge density. Effect of ionic strength (IS) showed that the resins with high net charge densities exhibited high adsorption capacities at high IS. Besides, dynamic binding capacity values achieved as high as 55 mg/mL for BSA and 143 mg/mL for LZM. Chromatographic experiments demonstrated favorable elution of bound proteins at NaCl lower than 370 mmol/L.

Effect of pore size on performance of monolithic tube chromatography of large biomolecules.

Effect of pore size on the performance of ion-exchange monolith tube chromatography of large biomolecules was investigated. Radial flow 1mL polymer based monolith tubes of different pore sizes (1.5, 2, and 6 μm) were tested with model samples such as 20 mer poly T-DNA, basic proteins, and acidic proteins (molecular weight 14000-670000). Pressure drop, pH transient, the number of binding site, dynamic binding capacity, and peak width were examined. Pressure drop-flow rate curves and dynamic binding capacity values were well correlated with the nominal pore size. While duration of the pH transient curves depends on the pore size, it was found that pH duration normalized on estimated surface area was constant, indicating that the ligand density is the same. This was also confirmed by the constant number of binding site values being independent of pore size. The peak width values were similar to those for axial flow monolith chromatography. These results showed that it is easy to scale up axial flow monolith chromatography to radial flow monolith tube chromatography by choosing the right pore size in terms of the pressure drop and capacity.

A megaporous material harbouring a peptide ligand for affinity IgG purification.

Common limitations of Protein A affinity chromatography include high adsorbent costs, ligand instability and possible ligand leakage. In this study, a short peptide with affinity for IgG was synthesized chemically and subsequently immobilized on a megaporous support. The support was prepared utilising the cryogel technique while the peptide-ligand was covalently immobilised via thiol-epoxy click chemistry. The cryogel support was chemically grafted to increase the number of reaction sites. This adsorbent was designated as "MP-Pep". Adsorption isotherms were employed to evaluate protein binding capacity. A maximum static binding capacity within the range of 30-60mg/mL was observed for T hIgG. This parameter compares well with other commercial and non-commercial adsorbents, as reported in the literature. As a control material, a Protein A grafted megaporous cryogel was synthesized. Dynamic binding capacity values were obtained by breakthrough analysis. The peptide cryogel showed a dynamic capacity value 9.0mg/mL in comparison to 9.7mg/mL in the case of the Protein A based adsorbent. The ratio of dynamic binding capacity to static binding capacity was 20%, indicating suboptimal product capture. However, the advantage of MP-Pep lies in its cost-effective preparations while maintaining a reasonable binding capacity for the targeted product. The presence of cooperative effects during protein binding could also represent an advantage during the processing of a feedstock containing a product in high concentration.

Grafting glycidyl methacrylate to Sepharose gel for fabricating high-capacity protein anion exchangers

To develop ion exchangers of high protein adsorption capacity, we have herein introduced atom transfer radical polymerization (ATRP) method to graft glycidyl methacrylate (GMA) onto Sepharose FF gel. GMA-grafted Sepharose FF resins of four grafting densities and different grafting chain lengths were obtained by adjusting reaction conditions. The epoxy groups on the grafted chains were functionalized by modification with diethylamine (DEA), leading to the fabrication of Sepharose-based anion exchangers of 14 different grafting densities and/or grafting chain lengths. The resins were first characterized for the effects of grafting density, chain length and ionic strength on pore sizes by inverse size exclusion chromatography. Then, the resins were evaluated by adsorption equilibria of bovine serum albumin (BSA) as a function of ionic capacity (IC) (chain length) at individual grafting densities. It was observed that at each grafting density there was a specific IC value (chain length) that offered the maximum equilibrium capacity. Of the resins with maximum values at individual grafting densities, the resin of the second grafting density with an IC value of 330mmol/L (denoted as FF-Br2-pG-D330) showed the highest capacity, 264mg/mL, about two times higher than that of the traditional ungrafted resin Q Sepharose FF (137mg/mL). This resin also showed the most favorable uptake kinetics among the resins of similar IC values but different grafting densities, or of the same grafting density but different IC values. Effects of ionic strength showed that the capacities of FF-Br2-pG-D330 were much higher than Q Sepharose FF at a wide range of NaCl concentrations (0–200mmol/L), and the uptake rates of the two resins were similar in the ionic strength range. Therefore, the dynamic binding capacity values of BSA on FF-Br2-pG-D330 were much higher than Q Sepharose FF as demonstrated at different residence times and ionic strengths. Taken together, the research has proved the success in the fabrication of high-capacity protein anion exchangers by grafting GMA onto Sepharose gel.

Silica-based strong anion exchange media for protein purification

The main objective of our research was to develop silica-based, polymer-functionalized ion exchange materials for single-use bioprocess applications, with the ultimate goal of achieving maximal binding capacity for target proteins. Herein we report the utilization of Grace® wide pore silica gel and bonding the silica with cationic polymers. The strong anion exchange materials have been prepared by a two-step process involving initial bonding with two trimethoxysilanes and subsequent aqueous solution radical polymerization with quaternary ammonium ion containing monomers and an azo initiator. Using the binding capacities for bovine serum albumin (BSA), a model protein for the evaluation of the new materials, we optimized the processes with regard to the median pore size of the silica gel, as well as polymer composition and ratios, which were determined by reagent ratios and reaction conditions. The products were also characterized by both chemical and physical methods. It has been found that higher binding capacities are associated with lower ligand density and higher molecular weight for the attached polymers, with over 20% higher in both static and dynamic binding capacity values with the same amount of attached polymers. The advantages of a large pore size distribution and optimal median pore size for the base silica are discussed. Optimal pore size range of 500–1500Å and distribution of Span 90 for over 1.0 give the highest BSA binding capacities. Silica-based strong anion exchange materials showed excellent flow characteristics when packed into a column and were superior to commercial agarose-based strong anion exchange material with respect to dynamic binding capacity, elution of proteins, and baseline separation of a mixture of three model proteins.

High‐throughput process development methods for chromatography and precipitation of proteins: Advantages and precautions

Although high‐throughput process development methods using 96‐well microplate (MP) systems are promising and attractive, they still have several unclear problems in the practical applications. Several case studies for the purification process development by using manually operated 96‐well MP systems are presented. In the first example, the MPs with ion‐exchange monolith disks or ion‐exchange membranes were employed for linear gradient elution and breakthrough curve experiments. The data such as the peak salt concentration and dynamic binding capacity values obtained with the MP system were in good agreement with those by the LC system. In the second example, batch adsorption experiments with a 96‐well filter MP were performed for a protein A resin (particle)–antibody system in order to obtain the adsorption isotherms. The static binding capacities calculated from the isotherms were similar to those calculated from the LC system. In the last example, protein precipitation experiments by PEG were carried out for determining the solubility curve. It was found that the presence of dimer and aggregates affect the solubility curve measurement significantly. The proposed methods suggest powerful practical applications of high‐throughput process development for chromatography and precipitation process when the experiments are carefully designed and carried out.

Distributed pore model for bio-molecule chromatography

One of the main peculiarities in protein chromatography is that the adsorbing proteins and the adsorbent pores have comparable sizes. This has the consequence that the pore accessibility depends not only on the solute size but also on the loading conditions of the adsorbent because protein adsorption significantly reduces the size of the pores. A model that accounts for the pore size distribution of the stationary phase and for the pore shrinkage due to protein adsorption has been developed to describe mass transport and adsorption in the porous particles. This model has been shown to be equivalent to the general rate model (GRM) in the case of processes under highly diluted conditions with little adsorption. This implies that the model parameters determination follows the same procedure as for the classical GRM. The new pore model has been applied and compared to the GRM for the simulation of lysozyme breakthrough experiments and for the prediction of 5% dynamic binding capacity values solely based on static capacity measurements.

Effects of adsorbent properties on zone spreading in expanded bed chromatography.

The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100-700 microm, density = 1100-1700 kg/m3, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose). The scale down study with 0.8 cm diameter columns was also attempted. Pulse response curves were measured with vitamin B12 as a tracer [Residence time distribution RTD experiments], and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time. The HETP values for different types of packing media tested showed very similar values (0.5-1.0 cm), which did not depend on the flow-rate or the column diameter (0.8-2.6 cm). Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC). The DBC values for EBC were similar to those for FBC. When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased. However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly.

Modeling of direct recovery of lactic acid from whole broths by ion exchange adsorption.

Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, Amberlite IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.

Retention behavior of very large biomolecules in ion-exchange chromatography

Hepatitis B virus surface antigen (HBsAg) particles were efficiently adsorbed (retained) on a Sulfate–cellulose (S–C) bead column, and then desorbed with sodium chloride solutions (0.5–3.0 M). The HBsAg particles were not efficiently retained onto either sulfopropyl–agarose (SP–A) or quaternary amine–agarose (Q–A) at pH 4.5, 6 and 8. The size-exclusion curve showed that proteins of molecular mass higher than ca. 20 000 cannot penetrate into the pores of S–C beads. The dynamic binding capacity (DBC) values of lysozyme (ca. 7 mg/ml-gel) and of γ-globulin (ca. 3 mg/ml gel) for S–C did not depend on the flow velocity while the DBC of γ-globulin for SP–A decreased sharply with an increase in flow velocity. These results indicated that very large molecules are adsorbed only onto the surface of S–C, which resulted in fast adsorption–desorption rates although the equilibrium adsorption capacity is lower than conventional porous gel beads. Because of the rapid adsorption rate, the DBC values of γ-globulin for S–C at high flow-rate regions are similar to those for SP–A. Bovine serum albumin was not adsorbed onto S–C. As this can not be explained by a simple electrostatic interaction mechanism, molecular recognition of S–C might be different from the agarose-based ion-exchange beads.