Molecular recognition and binding between human plasminogen Kringle 5 and α-chain of human complement component C3b by frontal chromatography and dynamics simulation.

The binding and molecular recognition between α-chain of human complement C3b (α-chain of C3b) and human plasminogen Kringle 5 (Kringle 5) were studied and explored by frontal chromatography and dynamics simulation in the combination of bio-specific technologies. The specific interaction between the α-chain of C3b and Kringle 5 was initially confirmed by ligand blot and ELISA (Kd = 4.243×10-6 L/mol). Furthermore, the binding determination conducted via frontal chromatography showed that the presence of a single binding site between them, with the binding constant of 2.98 × 105 L/mol. Then the molecular recognition by dynamics simulation and molecular docking showed that there were 9 and 13 amino acid residues respective in the Kringle 5 and α-chain of C3b directly implicated in the binding and the main stabilizing forces were electrostatic force (-55.99 ± 11.82 kcal/mol) and Van der Waals forces (-42.70 ± 3.45 kcal/mol). Additionally, a loop structure (65-71) in Kringle 5 underwent a conformational change from a random structure to an α-helix and a loop structure (417-425) in α-chain of C3b was closer to the molecular center, both of them were more conducive to the binding between them. Meanwhile, the involvement of the lysine binding site of Kringle 5 played an important role in the binding process. In addition, the erythrocyte-antibody complement rosette assay substantiated that the presence of Kringle 5 hindered the transportation of α-chain of C3b to antigen-antibody complex in a dose-dependent manner. These findings collectively indicated that the α-chain of C3b is very likely a receptor protein for Kringle 5, which provides a methodology for other similar investigations and valuable insights into expansion of the pharmacological effects and potential application of Kringle 5 in immune-related diseases.

Radiostrontium determination by combination of automated Sr isolation and "on-column" Cherenkov detection using chromatographic model.

A novel analytical solution of non-linear chromatography in case of parabolic isotherm for frontal analysis was obtained by combination of Cole-Hopf and Laplace transform. It was used for simulation of strontium capturing on chromatographic column with aim to improve quantitative determination of low-level 90Sr activities. From the experimentally determined breakthrough curves, the retention factor and the number of theoretical plates were calculated using the Glueckauf and Wenzel relations and by fitting the breakthrough curves for the linear isotherm using MatLab. These were used to simulate the breakthrough curves using a parabolic isotherm solution where the non-linear term of the isotherm was taken as a small negative deviation of the retention factor. On the base of theoretical prediction and experimental data, procedure for automated capturing of strontium on chromatographic column with specific dimension and off line "on-column" Cherenkov detection on commercial ultra low-level liquid scintillation counter was developed. It was shown that analytical solution for parabolic isotherm in comparison with solution for linear isotherm gives better prediction of mass of captured Sr on column filled with small amount of Sr resin and SuperLig®620 in case of elevated Sr concentration, even when non-linear effect is not obvious. The solution also makes it possible to predict the mass of resin required for strontium isolation at 100% yield under given conditions. Considering the limited dimensions of the column, and consequently small mass of the resin in them, it resulted in the low efficiency of the columns, which, however, did not affect the yield in real conditions of isolation. The results have shown that the yields achieved after isolation on SuperLig®620 from real samples are 100%. In addition, it is shown that captured 90Sr can be detected through 90Y ingrowth, on column filled with strontium specific resin, with Cherenkov detection efficiency of at least 50%. The efficiency may be enhanced to 60%, depending on parameters which can affect detection efficiency change (type of column, resin type, surrounding solution, etc.). The developed procedures enable quantitative determination of 90Sr in natural water samples with MDAC below 12 mBq l-1 and solid (soil and vegetation) samples with MDAC below 6 Bq kg-1 within 2-3 days. The proposed solution may easily be implemented in radiochemical laboratories where this type of analysis is routinely done within environmental monitoring or other purposes.

Resin structure impacts two-component protein adsorption and separation in anion exchange chromatography.

The influence of the resin structure, on the competitive binding and separation of a two-component protein mixture with anion exchange resins is evaluated using conalbumin and green fluorescent protein as a model system. Two macroporous resins, one with large open pores and one with smaller pores, are compared to a resin with grafted polymers. Investigations include measurements of single and two-component isotherms, batch uptake kinetics and two-component column breakthrough. On both macroporous resins, the weaker binding protein, conalbumin, is displaced by the stronger binding green fluorescent protein. For the large pore resin, this results in a pronounced overshoot and efficient separation by frontal chromatography. The polymer-grafted resin exhibits superior capacity and kinetics for one-component adsorption, but is unable to achieve separation due to strongly hindered counter-diffusion. Intermediate separation efficiency is obtained with the smaller pore resin. Confocal laser scanning microscopy provides a mechanistic explanation of the underlying intra-particle diffusional phenomena revealing whether unhindered counter-diffusion of the displaced protein can occur or not. This study demonstrates that the resin's intra-particle structure and its effects on diffusional transport are crucial for an efficient separation process. The novelty of this work lies in its comprehensive nature which includes examples of the three most commonly used resin structures: a small pore agarose matrix, a large-pore polymeric matrix, and a polymer grafted resin. Comparison of the protein adsorption properties of these materials provides valuable clues about advantages and disadvantages of each for anion exchange chromatography applications.

Sample displacement chromatography of monoclonal antibody charge variants and aggregates.

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5 pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35 mg of purified IgG fraction per milliliters of monolithic support with 75-80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.

Displacement to separate host-cell proteins and aggregates in cation-exchange chromatography of monoclonal antibodies.

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host-cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind-and-elute cation-exchange chromatography (CEX) polishing step. A variant of the bind-and-elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%-1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low-prevalence HCPs resulted in an effectively bimodal-like distribution of HCPs along the length of a multi-column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of low-prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography.

Human laminin α3 chain G1 domain is a receptor for plasminogen Kringle 5 on human endothelial cells by biological specificity technologies and molecular dynamic.

Human plasminogen Kringle 5 is known to pose a more potent anti-angiogenesis effect by inducing endothelial cell apoptosis. Our previous studies have identified the peptide IGNSNTL as a binding sequence of Kringle 5 using Ph.D.-7 phage display peptide library and enzyme-linked immunosorbent assay. Here, eleven proteins were screened and summarized by BLAST, laminin α3 chain G1 domain (LG1) was considered as the most potential receptor based on E value and domain function. The specific interaction of them was directly revealed through ligand blot and a strong concentration-dependent manner occurred between them (Ka 4.30 × 105 L mol-1) in frontal chromatography observation. Moreover, R10A/P83R substitution Kringle 5 decreased the affinity capacity to LG1. Furthermore, a remarkable conformational change from random coil3 to α helix and α1 helix to random coil were observed to the structural compactness and stability for LG1. Surface loops and coils also showed fluctuations up to some extent, giving the binding surface greater flexibility and correspondingly allowing for induced-fit binding, which was -23.87 kcal mol-1 of the free energy with electrostatic force as a main driver. Taken together, not only effective theoretical prediction and experiment validated that LG1 is receptor of Kringle 5, but also give an new perspective of the binding mechanism of Kringle 5 and its specific receptor and could facilitate the development of novel agent targeted toward pathologic angiogenesis.

Design space and robustness analysis of batch and counter-current frontal chromatography processes for the removal of antibody aggregates.

Increasing molecular diversity and market competition requires biopharmaceutical manufacturers to intensify their processes. In this respect, frontal chromatography on cation exchange resins has shown its potential to effectively remove aggregates. However, yield losses during the wash step need to be accepted in order to ensure robust product quality. In this work, we present a novel counter-current frontal chromatography process called Flow2, which uses inline dilution during an interconnected wash phase to allow high monomer recovery without contaminating the product pool with impurities. Its model-based design spaces under purity and yield constraints are compared with those corresponding to traditional batch processes in terms of size and process attributes yield and productivity. The Flow2 process shows the largest extent of feasible operating points independent of feed conditions. Thereby, it allows the implementation of higher ionic strength wash, thus widening the range of operating conditions resulting in yields above 95% compared to batch processes. Productivities of batch and counter-current processes are the same at short regeneration times and equal residence time. However, long regeneration times, while influencing the size of the Flow2 design space, are not detrimental for its productivity resulting in twice as high values as obtained for the batch process. Furthermore, process robustness is evaluated by the ability of the process to maintain the required product quality when subjected to process parameter perturbations. It is found that the Flow2 process is able to retain a larger design space associated also with higher yields showing its ability to improve process attributes without sacrificing robustness at the same time.

Process intensification by frontal chromatography: Performance comparison of resin and membrane adsorber for monovalent antibody aggregate removal.

Aggregates are amongst the most important product-related impurities to be removed during the downstream processing of antibodies due to their potential immunogenicity. Traditional operations use cation-exchange resins in bind-elute mode for their separation. However, frontal analysis is emerging as an alternative. In this study, a three-step process development for a membrane adsorber and a resin material is carried out, allowing the comparison between the stationary phases. Based on a screening study, optimal loading conditions are determined, which show that weak binding is favored on the membrane and strong binding on the resin. Transfer of these findings to breakthrough experiments shows that at 99% pool purity the yield is higher for the membrane, while the resin can be loaded twice as high, exceeding yields of 85%. For the investigated antibody and based on a given regeneration protocol, the productivity of the two phases is similar, ranging around 200 g/(L·h). Due to the higher loading, the resin requires about one-third less buffer than the membrane. Furthermore, the implementation of a wash step after loading allows to further increase yield by about 5%. In comparison to a generic bind-elute process, productivity and buffer consumption are improved by an order of magnitude.

Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates.

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates enabling much higher loadings. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molecular weight aggregates. The removal of mAb aggregates with Eshmuno® CP-FT resin using a continuous loading process was compared with six other commercially available strong CEX chromatography resins and found to correlate with their ionic densities, but not their mAb static binding capacities. The influence of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process was also investigated. Finally, the percentage of aggregates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process.

Breakthrough during air sampling with polyurethane foam: What do PUF 2/PUF 1 ratios mean?

Frontal chromatography theory is applied to describe movement of gaseous semivolatile organic compounds (SVOCs) through a column of polyurethane foam (PUF). Collected mass fractions (FC) are predicted for sample volume/breakthrough volume ratios (τ = VS/VB) up to 6.0 and PUF bed theoretical plate numbers (N) from 2 to 16. The predictions assume constant air concentrations and temperatures. Extension of the calculations is done to relate the collection efficiency of a 2-PUF train (FC1+2) to the PUF 2/PUF 1 ratio. FC1+2 exceeds 0.9 for PUF 2/PUF 1 ≤ 0.5 and lengths of PUF commonly used in air samplers. As the PUF 2/PUF 1 ratio approaches unity, confidence in these predictions is limited by the analytical ability to distinguish residues on the two PUFs. Field data should not be arbitrarily discarded because some analytes broke through to the backup PUF trap. The fractional collection efficiencies can be used to estimate air concentrations from quantities retained on the PUF trap when sampling is not quantitative.

Study on Binding Characteristics of Aminocarboxyl Chelating Ligands for Metal Ions / 分析化学

The feasibility of frontal chromatography for determining the complexation stability constant KML and total mole of binding site Λo was demonstrated by the accuracy and precision binding experiments between metal ions (Cu2+, Ni2+ and Co2+) and chelating ligand (IDA), in which R2>0.98 and RSD Asp>Glu;binding strength of metal ions for chelate ligands followed Cu2+>Ni2+>Co2+;and the binding effect with NaAc-HAc buffer was the best.In aqueous solution, quantum computing of binding energy (ΔE) and gibbs free energy (ΔG) between chelating ligand and metal ion was performed at the M06/6-311++G (d, p) level.According to ΔE and ΔG, the binding rules between chelating ligand and metal ion were predicted theoretically.These rules were basically in agreement with above experimental results.The present work provided effective method for studying on binding characteristics of metal ions for aminocarboxyl chelating ligands, thus exhibited a good foundation for improving the stability of immobilized metal affinity chromatographic column and solving the leakage of metal ions from the column in the process of competitive elution.

Evaluation of efficiency and trapping capacity of restricted access media trap columns for the online trapping of small molecules.

Restricted access media are generally composed from multi-modal particles that combine a size excluding outer surface and an inner-pore retention mechanism for small molecules. Such materials can be used for either online isolation and pre-concentration of target small molecules or removal of small molecule interferences from large macromolecules, such as proteins in complex biological matrices. Thus, they are considered as enhanced online solid-phase extraction materials. We evaluated the efficiency and trapping capacity of different semi-permeable surface restricted access media columns (C18 , C8 , and C4 inner pores) for four model small molecule compounds (dopamine hydrochloride, acetaminophen, 4-hydroxybenzoic acid, and diethyl phthalate) having variable physicochemical properties. We further studied the effect of mobile phase flow rate (0.25, 0.5, 1, and 2 mL/min) and pH, using 98:2 0.5% acetic acid in water/ methanol (pH 2.88) and 5 mM ammonium acetate in 98:2 water/methanol (pH 6.61) as mobile phases. Breakthrough curves generated using frontal analysis were analyzed to determine important chromatographic parameters specific for each of the studied compounds. Experimental determination of these parameters allowed selection of the most efficient trap column and the best loading mobile phase conditions for maximal solute enrichment and pre-concentration on restricted access media trap columns.

Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors.

Evaluation of sorbent materials for the sampling and analysis of phosphine, sulfuryl fluoride and methyl bromide in air.

Phosphine (PH3), sulfuryl fluoride (SO2F2) and methyl bromide (CH3Br) are highly toxic chemical substances commonly used for fumigation, i.e., pest control with gaseous pesticides. Residues of fumigation agents constitute a health risk for workers affected, and therefore accurate methods for air sampling and analysis are needed. In this study, three commercial adsorbent tubes; Carbosieve SIII™, Air Toxics™ and Tenax TA™, were evaluated for sampling these highly volatile chemicals in air and their subsequent analysis by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The breakthrough volume (BTV) of each fumigant was experimentally determined on the different adsorbents at concentrations at or above their permissible exposure limits, using a method based on frontal chromatography of generated fumigant atmospheres. Carbosieve SIII™, a molecular sieve possessing a very high specific area, proved to be a better adsorbent than both Air Toxics™ and Tenax TA™, resulting in at least a 4-fold increase of the BTV50%. BTV50% for Carbosieve SIII™ at 20°C was measured as 4.7L/g, 5.5L/g and 126L/g for phosphine, sulfuryl fluoride and methyl bromide, respectively, implying safe sampling volumes of 1.9L, 2.2L and 50L, respectively, for a commercial tube packed with 800mg Carbosieve SIII™. The temperature dependence of BTV was strong for Carbosieve SIII™, showing a reduction of 3-5%/°C in breakthrough volume within the range -20 to 40°C. Furthermore, although Carbosieve SIII™ reportedly has a higher affinity for water than most other adsorbents, relative humidity had only a moderate influence on the retention capacity of phosphine. Overall, the applicability of Carbosieve SIII™ adsorbent sampling in combination with TD-GC-MS analysis was demonstrated for highly volatile fumigants.

Adsorption of the anionic surfactant sodium dodecyl sulfate on a C18 column under micellar and high submicellar conditions in reversed-phase liquid chromatography.

Micellar liquid chromatography makes use of aqueous solutions or aqueous-organic solutions containing a surfactant, at a concentration above its critical micelle concentration. In the mobile phase, the surfactant monomers aggregate to form micelles, whereas on the surface of the nonpolar alkyl-bonded stationary phases they are significantly adsorbed. If the mobile phase contains a high concentration of organic solvent, micelles break down, and the amount of surfactant adsorbed on the stationary phase is reduced, giving rise to another chromatographic mode named high submicellar liquid chromatography. The presence of a thinner coating of surfactant enhances the selectivity and peak shape, especially for basic compounds. However, the risk of full desorption of surfactant is the main limitation in the high submicellar mode. This study examines the adsorption of the anionic surfactant sodium dodecyl sulfate under micellar and high submicellar conditions on a C18 column, applying two methods. One of them uses a refractive index detector to obtain direct measurements of the adsorbed amount of sodium dodecyl sulfate, whereas the second method is based on the retention and peak shape for a set of cationic basic compounds that indirectly reveal the presence of adsorbed monomers of surfactant on the stationary phase.

Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7µM), verapamil (0.6 vs. 0.7µM) and prazosin (0.099 vs. 0.033µM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

New trends in LC protein ligand screening.

The diversity of small molecules available to produce truly innovative drugs associated with the wealth of known biological targets calls for key strategies in protein ligand screening. This review encompasses the recently developed bioaffinity-based strategies. A critical view of the use of zonal, frontal, and nonlinear chromatography with immobilized proteins is given. The association of these elution modes with the ligand fishing method, which uses nanomagnetics particles, is also addressed. A series of applications and how these new screening strategies can be used to determine the function, affinity, and activity parameters of proteins is discussed.