Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.     

Adsorption and diffusion of CO2 and CH4 in covalent organic frameworks: an MC/MD simulation study

Covalent organic frameworks (COFs) are a promising gas separation material which have been developed recently. In this work, we have used grand canonical Monte Carlo (GCMC) and molecular dynamics (MD) simulations to investigate the adsorption and diffusion properties of CO₂ and CH₄ in five recent synthesised COF materials. We have also considered the properties of amino-modified COFs by adding –NH₂ group to the five COFs. The adsorption isotherm, adsorption/diffusion selectivity, self/transport diffusion coefficients have been examined and discussed. All of the five COFs exhibit promising adsorption selectivity which is higher than common nanoporous materials. An S-shaped adsorption isotherm can be found for CO₂ instead of CH₄ adsorption. The introduction of –NH₂ group is effective at low pressure region (<200?kPa). The diffusion coefficients are similar for TS-COFs but increase with the pore size for PI-COFs, and the diffusion coefficients seem less dependent on the –NH₂ groups.     

Enhancing protein capacity of rigid macroporous polymeric adsorbent.

A macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) resin was synthesized and modified with diethylamine to yield an anion-exchange resin suitable for protein adsorption. Efforts were made to enhance protein ion exchange capacity of the resin by investigating the copolymer composition. Different synthesis recipes were attempted, and the resultant resins were characterized by measuring the specific surface area and the adsorption ability using bovine serum albumin (BSA) as a model protein. The intraparticle pore size distribution measured by mercury porosimetry showed that the pores in the range of 40-120 nm took 75% of the total pore volume, indicating that the ion exchanger was favorable for protein adsorption. BSA capacity obtained with an appropriate recipe was as high as 78.6 mg/g wet resin or 50 mg/mL packed volume, which was higher than the capacities of some commercially available ion exchangers. Moreover, by using a pore diffusion model, the effective pore diffusivity of BSA was found to be 5.5 x 10(-12) m(2)/s, similar to those in the commercial ion exchangers.     

Adsorption kinetics and equilibria of bovine serum albumin on rigid ion-exchange and hydrophobic interaction chromatography matrices in a stirred cell

Rigid adsorbents have advantages over soft gel media for downstream processing of proteins. The adsorption of bovine serum albumin (BSA) has been investigated on a rigid adsorbent based on a wide-pore, hydrophilically coated, silica-gel matrix. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction chromatography) and particle size have been studied on the physical properties of the adsorbent and on the adsorption equilibria and adsorption kinetics. The rates of adsorption of BSA have been measured in a stirred cell and are found to be satisfactorily described by a two-step theoretical model, in which the mass transfer involves a pore diffusion resistance and an extra-particle film resistance. On the anion exchanger, the effective pore diffusivity decreases substantially with increasing protein concentration, approximately halving as the initial concentration rises from 0.7 to 2g/l. In the hydrophobic interaction chromatography medium, the pore diffusivity is less sensitive to protein concentration and is also reduced by a factor of about 4 by aggregation of the protein. Effective pore diffusivities with the "wide-pore" silica adsorbents in anion-exchange form are 36-94 times lower than the diffusivity in free solution and are comparable with the lower of the wide range of values published for soft gels.     

Competitive adsorption of binary mixture of <Emphasis Type="Italic">Leptospirillum ferriphilum</Emphasis> and <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> onto pyrite

Leptospirillum ferriphilum and Acidithiobacillus caldus are two important acidophilic microorganisms involved in iron and sulfur oxidation during bioleaching. Cell adsorption to mineral surfaces is important for the direct leaching or contact leaching of minerals. In this study, we report the competitive adsorption of binary mixtures of L. ferriphilum LF-104 and A. caldus MTH-04 onto pyrite surfaces. The Langmuir adsorption parameter (C_Am) indicated that these two bacteria underwent competitive adsorption to pyrite. Real-time quantitive PCR was used to quantify the relative amounts of L. ferriphilum and A. caldus adsorbed onto the surfaces of pyrite following exposure to a mixture of these two organisms. The adsorption of L. ferriphilum was not affected by A. caldus. However, adsorption of A. caldus was greatly affected by the presence of L. ferriphilum. Zeta-potential measurements and FT-IR spectroscopic studies showed that L. ferriphilum had a higher electrostatic attraction towards pyrite when compared to A. caldus. Based on the above results, we propose a competitive adsorption model to explain the mechanism by which L. ferriphilum and A. caldus compete in their adsorption to pyrite, although L. ferriphilum dominated in the competitive adsorption process. This work provides a better understanding of the adsorption behavior of mixed microbial populations onto mineral surfaces.     

Insulin adsorption into porous charged membranes: Effect of the electrostatic interaction

Insulin adsorption into a series of porous charged membranes was investigated by batch adsorption experiments, and the experimental results were analyzed by the homogeneous diffusion model. The membranes used in this study were prepared by pore‐surface modification of porous poly(acrylonitrile) (PAN) membranes by grafting with weak acidic and basic functional groups. The amount of insulin adsorbed into the membrane was determined from the material balance of insulin. The insulin partition coefficient K between the membrane and solution was estimated from the equilibrium adsorption amount, and the effective diffusion coefficient D was estimated by matching the model with the experimental data as a fitting parameter. The dependence of K and D on the charge properties of the insulin and membrane is observed and discussed. The partition coefficient K increased when the insulin and the membrane carried opposite charges, on the other hand, the effective diffusion coefficient D was reduced. These results indicate that the electrostatic interaction between the insulin and the membranes played an important role in the insulin adsorption. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Competitive adsorption of human fibrinogen on an amorphic quartz surface

The kinetics of adsorption of fibrinogen on the surface of amorphous quartz from binary solutions containing human serum albumin and gamma-globulin was studied by the method of fluorescence of total internal reflection. A model of energetically nonhomogeneous interactions in the surface/protein system was used to explain the mechanism of competitive adsorption of proteins.     

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K_p value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425–434, 2017     

Preparation of a novel hydrophobic affinity cryogel for adsorption of lipase and its utilization as a chromatographic adsorbent for fast protein liquid chromatography

In this study, we have prepared a hydrophobic cryogel for the chromatographic separation of lipase from its aqueous solutions including single protein and protein mixture and also Yarrowia lipolytica cell extract. N‐methacryloyl‐(l )‐phenylalanine methyl ester was used as a monomer to provide the hydrophobic character to the prepared cryogels. The highest adsorption capacity was observed at pH 5.0 at 0.5 mL min^?1 flow rate. The chromatographic separation of lipase was achieved from a binary mixture of lipase:bovine serum albumin (BSA) and lipase:lysozyme, and was also achieved from triple‐mixture of lipase:lysozyme:BSA by using fast protein liquid chromatography. Finally, lipase purification was performed from Yarrowia lipolytica cell extract used as a natural source. These studies have shown that the hydrophobic cryogel has good chromatographic performance for the separation and purification of lipase not only from aqueous solution, but also from cell extract as a natural source of lipase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:376–382, 2014     

Computer Simulation of Isothermal Mass Transport in Graphite Slit Pores

Abstract

Results are presented from a simulation study of the mass transport of oxygen and nitrogen through graphite slit pores. The work is motivated by an attempt to understand the molecular origins of the kinetic selectivity displayed when air is separated into its major components using pressure swing adsorption. A combination of non-equilibrium molecular dynamics (NEMD), equilibrium molecular dynamics (EMD) and grand canonical Monte Carlo methods has been employed in our study to extract the maximum information. Transport diffusivities, self-diffusivities, permeabilities and Darken thermodynamic factors have been calculated as a function of pore width and temperature for pure component oxygen and nitrogen. In addition, new EMD simulation data for an 80:20 mixture of nitrogen and oxygen is reported, including a direct calculation of the Stefan-Maxwell coefficients. The results are discussed in terms of the oxygen selectivity and the possible mechanisms, which increase or decrease this quantity.

We find that the pore width behaviour of the diffusion coefficients consists of three distinct regimes: a regime at larger pore widths in which single component diffusion coefficients are largely independent of pore width, an optimum pore width at which both diffusivities increase substantially but the slit pore is selective towards nitrogen, and a regime at very low pore widths at which the diffusivities decrease sharply, but the slits are selective towards oxygen. The mechanism behind each of these regimes is discussed in terms of “entropic” effects and potential barrier heights.

We have also found that permeability selectivity is substantially reduced in a mixture of the two gases with a composition similar to that of air. Cross diffusion coefficients in the mixture have been calculated and shown to be non-negligible.     

Apolipoprotein A-I(Milano) anion exchange chromatography: mass transfer and adsorption kinetics

The mass transfer and adsorption kinetics of self-associating apolipoprotein A-I(Milano) (apoA-I(M)) was investigated for the two anion exchangers Q-Sepharose-HP and Macro-Prep-HQ. At high salt where no protein binding occurs and without urea, mass transfer was controlled by hindered pore diffusion of multiple associated forms for both materials. Adding urea suppressed self-association, but resulted in higher viscosity and caused unfolding. As a consequence, the effective diffusivity decreased as urea was added and was greater for the larger pore Macro-Prep-HQ resin. At low salt, under strong binding conditions, the adsorption kinetics followed a more complex mechanism. In this case, the kinetics was very slow for both stationary phases up to 2 M urea. However, at higher urea concentrations, the adsorption kinetics for the smaller pore Q-Sepharose-HP matrix became much faster, suggesting a transition from pore- to surface-dominated diffusion. Microscopic observations confirmed that different transport mechanisms were in play below and above 2 M urea, which marked the approximate boundary above which self-association was suppressed and unfolding occurred. The net result was enhanced uptake kinetics at high urea concentrations (e.g., 4 M) where protein unfolding is thought to lead to a more flexible structure that can reptate along the pore surface. Although the observed enhancement was dependent on the pore size and, thus, the surface area of the resin, it was not limited to apoA-I(M). BSA showed a similar trend as a function of urea when its disulfide bonds were reduced.     

1,3‐Propanediol adsorption on a cation exchange resin: Adsorption isotherm,thermodynamics, and mechanistic studies

1,3‐Propanediol (1,3‐PD) is widely used in cosmetics, foods, antifreezes, and polyester. A low‐cost cation exchange resin, 001×7 H‐form resin, was selected for 1,3‐PD adsorption obtained from microbial fermentation of crude glycerol. The thermodynamics and kinetics of adsorption were studied. To identify the characteristics of the adsorption process, the influence of 1,3‐PD concentration, temperature, and resin particle diameters was studied. The temperature dependence of the adsorption equilibrium in the range of 288 to 318 K was observed to satisfy the Langmuir isotherm well. The thermodynamic parameters, adsorption enthalpy, entropy, and Gibbs free energy, were determined as 36.2 kJ·moL^?1, 160 mol^?1·K^?1, and ?11.2 kJ·moL^?1, respectively, which indicated that the adsorption was spontaneous and endothermic. The adsorption kinetics was accurately represented by the shell progressive model and indicated that the particle diffusion was the rate‐limiting step. Based on the kinetic simulation, the rate constant of exchange (k₀), order reaction (α), and the apparent activation energy reaction (E_a) were obtained as 3.11×10^?3, 0.644, and 11.5 kJ·moL^?1, respectively. This kinetic and thermodynamic analysis of 1,3‐PD recovery presented in this article is also applicable for the separation of other polyols by resin adsorption, which will promote value‐added utilization of glycerol.     

2-Mercapto-5-benzimidazolesulfonic acid: an effective multimodal ligand for the separation of antibodies

The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0-5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step.     

用超大孔树脂吸附和低乙醇浓度洗脱提纯异黄酮的研究

传统大孔吸附树脂吸附分离中草药有效成分通常需要高浓度的有机溶剂作为洗脱剂,在大规模生产中具有安全隐患和对环境的污染。本文以异黄酮为目标产物,考察了8种不同骨架、不同孔径的树脂对异黄酮的吸附分离性能,其中包括本实验室自制的孔径为1000nm的超大孔树脂。实验结果表明,与传统大孔树脂吸附层析法相比,超大孔树脂吸附层析不仅工艺简单、快速而且大幅度降低了有机溶剂的使用量。洗脱时,乙醇浓度由70％降低到20％。     

Anticorrosion mechanism of Cr-doped nickel-base alloy in Br/O environment: a DFT study

ABSTRACT

Density functional theory was used to investigate the anticorrosion mechanism of Cr-doped nickel-base alloy in Br/O environment. The adsorption properties including adsorption energy, bond length and electron structure of Br/O on Ni and Cr-doped Ni surfaces have been investigated. We find that the O atom has bigger adsorption activity than the Br atom. The oxide film can be preferentially formed on the Cr-doped nickel-base alloy surface under the coexistence of Br and O atoms. By exploring the interactions between Br and NiO (111) or Cr₂O₃ (100) surfaces, we demonstrate that oxide films could prevent Br from erosion. Results indicate that Cr₂O₃ has stronger corrosion resistance than NiO. The corrosion resistance mechanism of Cr-doped nickel-base alloy is summarised into two processes, i.e. competitive adsorption and adsorption inhibition.     

Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders

We compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix.     

Adsorption of rutin with a novel β-cyclodextrin polymer adsorbent: Thermodynamic and kinetic study

The adsorption properties toward rutin of a cyclodextrin polymer adsorbent CroCD-TuC 3 have been studied. The adsorption capacity is reduced as temperature and pH of solution rises, but increases with the increase of solvent polarity. Compared with Sephadex? G-15 dextran gel beads, CroCD-TuC 3 shows dramatically higher isosteric enthalpy due to a significant contribution of rutin/β-cyclodextrin inclusion complex formation in CroCD-TuC 3 skeleton. A highlight in our study is that the pore diffusion model has been employed to describe the mass transfer inside the adsorbent pores. It reveals that the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The effective pore diffusivity of rutin in CroCD-TuC 3 calculated is much lower than the diffusivity in diluted solution. The pore diffusion model is an available tool to investigate the profile of mass transfer inside the pores, and provides an effective method to describe adsorption kinetics.     

The role of lactic acid adsorption by ion exchange chromatography

Background

The polyacrylic resin Amberlite IRA-67 is a promising adsorbent for lactic acid extraction from aqueous solution, but little systematic research has been devoted to the separation efficiency of lactic acid under different operating conditions.

Methodology/Principal Findings

In this paper, we investigated the effects of temperature, resin dose and lactic acid loading concentration on the adsorption of lactic acid by Amberlite IRA-67 in batch kinetic experiments. The obtained kinetic data followed the pseudo-second order model well and both the equilibrium and ultimate adsorption slightly decreased with the increase of the temperature at 293–323K and 42.5 g/liter lactic acid loading concentration. The adsorption was a chemically heterogeneous process with a mean free energy value of 12.18 kJ/mol. According to the Boyd^_plot, the lactic acid uptake process was primarily found to be an intraparticle diffusion at a lower concentration (<50 g/liter) but a film diffusion at a higher concentration (>70 g/liter). The values of effective diffusion coefficient D_i increased with temperature. By using our Equation (21), the negative values of ΔG° and ΔH° revealed that the adsorption process was spontaneous and exothermic. Moreover, the negative value of ΔS° reflected the decrease of solid-liquid interface randomness at the solid-liquid interface when adsorbing lactic acid on IRA-67.

Conclusions/Significance

With the weakly basic resin IRA-67, in situ product removal of lactic acid can be accomplished especially from an open and thermophilic fermentation system without sterilization.     

SP-B and SP-C alter diffusion in bilayers of pulmonary surfactant

The hydrophobic proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface by an unknown mechanism. We tested the hypothesis that these proteins accelerate adsorption by disrupting the structure of the lipid bilayer, either by a generalized increase in fluidity or by a focal induction of interfacial boundaries within the bilayer. We used fluorescence recovery after photobleaching to measure diffusion of nitrobenzoxadiazolyl-dimyristoyl-phosphatidylethanolamine between 11 and 54 degrees C in multilayers containing the complete set of lipids and proteins in calf lung surfactant extract (CLSE), or the complete set of neutral and phospholipids without the proteins. Above 35 degrees C, Arrhenius plots of diffusion were parallel for CLSE and neutral and phospholipids, but shifted to lower values for CLSE, suggesting that the proteins rigidify the lipid bilayer rather than producing the proposed increase in membrane fluidity. The slopes of the Arrhenius plots for CLSE were steeper below 35 degrees C, suggesting that the proteins induce phase separation at that temperature. The mobile fraction fell below 27 degrees C, consistent with a percolation threshold of coexisting gel and liquid-crystal phases. The induction of lateral phase separation in CLSE, however, does not correlate with apparent changes in adsorption kinetics at this temperature. Our results suggest that SP-B and SP-C accelerate adsorption through a mechanism other than the disruption of surfactant bilayers, possibly by stabilizing a high-energy, highly curved adsorption intermediate.