Frontal Chromatography Research Articles

Exploring the influence of copper precursors and solvents on catalyst performance in the hydrogenation of hydroxyacetone to 1,2-Propanediol

This study proposes the impregnation of silica gel with copper acetate dissolved in water as a valid alternative for the preparation of catalysts for hydrogenation of oxygenates. Copper precursors demonstrated a significant impact on the catalytic properties of the samples here prepared. Moreover, the use of copper acetate in combination with water as impregnation solvent led to the highest activity among the samples presented in this work, suggesting a role of solvent polarity. Characterization by TPR allowed the identification of an unexpected consumption of hydrogen per copper atom, which was associated to the ability to localize hydrogen. The presence of Cu+ ions in the samples was investigated by CO chemisorption, N2O reactive frontal chromatography and XPS. However, no evidence of its impact on the activity was established.

Optimizing Antimony Speciation Analysis via Frontal Chromatography-ICP-MS to Explore the Release of PET Additives.

Antimony (Sb) contamination poses significant environmental and health concerns due to its toxic nature and widespread presence, largely from anthropogenic activities. This study addresses the urgent need for an accurate speciation analysis of Sb, particularly in water sources, emphasizing its migration from polyethylene terephthalate (PET) plastic materials. Current methodologies primarily focus on total Sb content, leaving a critical knowledge gap for its speciation. Here, we present a novel analytical approach utilizing frontal chromatography coupled with inductively coupled plasma mass spectrometry (FC-ICP-MS) for the rapid speciation analysis of Sb(III) and Sb(V) in water. Systematic optimization of the FC-ICP-MS method was achieved through multivariate data analysis, resulting in a remarkably short analysis time of 150 s with a limit of detection below 1 ng kg-1. The optimized method was then applied to characterize PET leaching, revealing a marked effect of the plastic aging and manufacturing process not only on the total amount of Sb released but also on the nature of leached Sb species. This evidence demonstrates the effectiveness of the FC-ICP-MS approach in addressing such an environmental concern, benchmarking a new standard for Sb speciation analysis in consideration of its simplicity, cost effectiveness, greenness, and broad applicability in environmental and health monitoring.

Ammonia and toluene oxidation: Mutual activating effect of copper and cerium on catalytic efficiency

Copper and cerium containing hydrotalcite-like precursors Cux-Cey-Mg-Al were synthesized by mutual co-precipitation followed by calcination at 800 °C. The obtained mixed metal oxides were studied as catalysts for selective ammonia oxidation (NH3-SCO) and toluene combustion. Various techniques, such as temperature-programmed reduction with hydrogen, temperature-programmed desorption of toluene, scanning electron microscopy and reactive frontal chromatography were used to characterize the catalysts. Series 800-Cux-Ce2.5 showed the highest catalytic efficiency in both reactions, allowing complete NH3 and C7H8 conversion below 350 °C and 500 °C, respectively. CeO2 crystallites (12–18 nm), as well as dispersed CuO oxide forms were found in the most active samples. Both phases affect pollutants conversion, however, for ammonia oxidation the occurrence of Cu2+ is essential, while for toluene oxidation, the formation of Ce4+ is sufficient. Over the most active sample (800-Cu5-Ce2.5) the complete conversion in mutual oxidation of NH3 and toluene was achieved below 450 °C.

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.

A high-throughput, straightforward procedure for biomonitoring organomercury species in human hair

Mercury is a pervasive and concerning pollutant due to its toxicity, mobility, and tendency to biomagnify in aquatic and terrestrial ecosystems. Speciation analysis is crucial to assess exposure and risks associated with mercury, as different mercury species exhibit varying properties and toxicities. This study aimed at developing a selective detection method for organic mercury species in a non-invasive biomonitoring matrix like human hair. The method is based on frontal chromatography (FC) in combination with inductively coupled plasma mass spectrometry (ICP-MS), using a low pressure, homemade, anion exchange column inserted in a standard ICP-MS introduction system, without requiring high-performance liquid chromatography (HPLC) hyphenation. In addition to the extreme simplification and cost reduction of the chromatographic equipment, the proposed protocol involves a fast, streamlined and fully integrated sample preparation process (in contrast to existing methods): the optimized procedure features a 15-min ultrasonic assisted extraction procedure and 5 min analysis time. Consequently, up to 100 samples could be analyzed daily, making the method highly productive and suitable for large-scale screening programs in public and environmental health. Moreover, the optimized procedure enables a limit of detection (LOD) of 5.5 μg/kg for a 10 mg hair microsample. All these features undeniably demonstrate a significant advancement in routine biomonitoring practices. To provide additional evidence, the method was applied to forty-nine human hair samples from individuals with varying dietary habits successfully finding a clear correlation between methylmercury levels (ranging from 0.02 to 3.2 mg/kg) in hair and fish consumption, in line with previous literature data.

Sampling efficiency of a polyurethane foam air sampler: Effect of temperature

Effective monitoring of atmospheric concentrations is vital for assessing the Stockholm Convention's effectiveness on persistent organic pollutants (POPs). This task, particularly challenging in polar regions due to low air concentrations and temperature fluctuations, requires robust sampling techniques. Furthermore, the influence of temperature on the sampling efficiency of polyurethane foam discs remains unclear. Here we employ a flow-through sampling (FTS) column coupled with an active pump to collect air samples at varying temperatures. We delved into breakthrough profiles of key pollutants, such as polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs), and organochlorine pesticides (OCPs), and examined the temperature-dependent behaviors of the theoretical plate number (N) and breakthrough volume (VB) using frontal chromatography theory. Our findings reveal a significant relationship between temperature dependence coefficients (KTN, KTV) and compound volatility, with decreasing values as volatility increases. While distinct trends are noted for PAHs, PCBs, and OCPs in KTN, KTV values exhibit similar patterns across all chemicals. Moreover, we establish a binary linear correlation between log (VB/m3), 1/(T/K), and N, simplifying breakthrough level estimation by enabling easy conversion between N and VB. Finally, an empirical linear solvation energy relationship incorporating a temperature term is developed, yielding satisfactory results for N at various temperatures. This approach holds the potential to rectify temperature-related effects and loss rates in historical data from long-term monitoring networks, benefiting polar and remote regions.

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.

Design of frontal chromatography separation of 1-phenylethanol and acetophenone using a hydrophobic resin

Adsorption and chromatographic processes havegood potential in the purification of natural aromas produced via biocatalytic routes. This work deals with the recovery of 1-phenylethanol (PE) from mixtures containing different amounts of acetophenone (AP). Amberlite FPX68, an industrial adsorbent with high selectivity with respect to acetophenone, was chosen for the separation of PE and AP by frontal chromatography. Small-scale experiments were performed for two different feed compositions and various flow rates and bed lengths. Using the target purity of 99 %, promising yields close to 90 % were obtained. The breakthrough curves were fitted with the equilibrium dispersive model, which provided HETP values under the different conditions. The obtained dependences of HETP on flow velocity complied with the Van Deemter equation. The contributions of individual phenomena responsible for adsorption zone broadening were analyzed. The experimental and modelling results obtained for the small-scale column became the basis for the scale-up of frontal chromatography separation of PE with an annual capacity up to 500 kg.

Protein adsorption and separation with monomodal and multimodal anion exchange chromatography resins. Part II. Mechanisms of protein aggregation on the chromatographic surface

AbstractBACKGROUNDProteins on chromatographic surfaces can undergo conformational changes, leading to aggregate formation. Such effects have been reported previously for hydrophobic interaction and cation exchange chromatography. In Part I of this work, we examined protein adsorption behavior on monomodal (Nuvia HP‐Q) and multimodal anion exchangers (Nuvia aPrime and Capto Adhere), observing that aggregates are also formed on these multimodal resins. In this work we examine the underlying mechanisms and the conditions that affect aggregate formation.RESULTSIncubating the multimodal anion exchangers with pure bovine serum albumin monomer at low ionic strength results in the formation of dimer and higher‐order oligomers (up to hexamers) to an extent that depends on the incubation time. The percentage of total protein initially bound that is desorbed decreased from >85% with a 5 min incubation to <40% with 24 h incubation at pH 7.0, indicating that additional irreversibly bound species are formed on the resin surface. Load buffers leading to stronger binding (higher pH and/or lower salt) and higher temperatures (32 °C) result in more extensive aggregation. Minimal aggregation occurs, instead, for Nuvia HP‐Q.CONCLUSIONBSA oligomers are formed directly on the surface of the multimodal resins studied and are eluted as discrete species at increasing salt concentrations. Aggregate formation is promoted by the ligand hydrophobicity and affects the separation of monomer–dimer mixtures by frontal chromatography, as the ability of the dimer in the feed to displace the adsorbed protein is impaired by gradual conversion of the monomer to stronger bound species. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Protein adsorption and separation with monomodal and multimodal anion exchange chromatography resins. Part I. Multicomponent adsorption properties and frontal chromatography

AbstractBACKGROUNDMultimodal ion exchangers, containing ligands combining charged and hydrophobic moieties, exhibit enhanced separation compared to monomodal ion exchangers under low loading conditions. Commercial applications often require high protein loads and such conditions have not been investigated extensively, especially for multimodal anion exchangers. This work compares protein adsorption and separation by frontal chromatography for a commercial monomodal (Nuvia HP‐Q) and two multimodal anion exchangers (Nuvia aPrime and Capto Adhere) using bovine serum albumin (BSA) monomer and dimer as a model.RESULTSGreater dimer–monomer selectivity is observed for Nuvia aPrime and Capto Adhere than for Nuvia HP‐Q at low protein loads. However, binary adsorption isotherms at high protein loads show higher binding capacity and greater selectivity for Nuvia HP‐Q, despite having the same ligand density. Higher salt concentrations reduce the binding strength in all cases, but this reduction is less pronounced for the multimodal resins. As a result, selectivity increases for the latter at higher salt concentrations. Confocal laser scanning microscopy shows that, during co‐adsorption, displacement of the monomer by the dimer occurs for each resin. However, displacement becomes very slow for the multimodal resins during sequential adsorption. The multimodal resins give poorer separation by frontal chromatography, accompanied by low recovery and substantial on‐column formation of protein aggregates.CONCLUSIONThe selectivity of multimodal anion exchangers for separation of the model proteins studied varies with salt concentration and protein load. At high protein loads, multimodal resins can result in on‐column aggregate formation negatively impacting the kinetics of competitive binding and separation. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

A model for purification of by-products in wood extracts based on frontal chromatography

ABSTRACT The present work consists of the development of a methodology based on reverse-phase frontal chromatography to separate and purify different fractions of wood extract. Equilibrium isotherms of each compound were first determined for 9 ethanol: water concentrations. A model was developed to simulate the profiles obtained by frontal chromatography. The fitting of simulated and experimental profiles led to the validation of the model and to the fact that the studied compounds present linear isotherms even in high concentration ranges. Simulation can therefore be carried out to optimize separations performances. Three purification strategies were considered: (a) separation of the two major classes of families, (b) separation of both Hydroxymatairesinol isomers, and (c) separation of each isomer. The simulation showed a high dependency on ethanol concentration and the best condition for optimizing the load volume was at the lower ethanol concentration (3 v.v.%). For a 250 × 4.6 mm analytical column, the simulation showed that volumes of 6021.0 mL (a), 450.2 mL (b) and 127.9 mL (c) could be loaded. Scale-up criteria are then briefly discussed for large-scale production.

Physical selectivity on isotopologues of gaseous alkanes by shale pore network: Evidence from dynamic adsorption process of natural gas

In this study, frontal chromatography experiments have been conducted with hydrocarbon gases (methane and ethane) on selected stationary phases (quartz, 3 Å, 5 Å and 13X molecular sieves, activated carbon, coal and shale) to study compositional and carbon isotope fractionation during adsorption process. The results show that the diffusion process cannot leads to obvious isotopic fractionation larger than ∼1.5 per mil in a relatively short distance. However, carbon isotope fractionation varies in different adsorption matrix. Gas molecules transporting in a relatively short distance can cause the isotopically light molecules weakly enriched in the front part of the alkane gases flows. The pore size distribution acts as the leading factor influencing the isotopic fractionation during hydrocarbon gases adsorption. In adsorption matrix with relatively low pore radius (i.e., activated carbon, 5 Å molecular sieve and coal), the isotopically light molecules can occupy the adsorption site preferentially and almost cannot be replaced due to the strong adsorption force. Hence, isotopically heavy molecules flow out of the system preferentially. To contrast, in adsorption matrix with relatively high pore radius (i.e., 13X molecular sieve and shale), the isotopically light molecules can occupy the adsorption site and desorb preferentially. Hence, isotopically light molecules flow out of the system preferentially after repeated adsorption and desorption. In addition, physical selectivity on isotopologues of gaseous alkanes by pore network also can occur in natural shale gas system. The Niutitang shale mainly is contributed by micropore. Whereas, Wufeng-Longmaxi shale mainly is contributed by mesopore. In general, initial adsorbed gases (residual gases) in natural shale samples from Niutitang shale were of more negative isotopic compositon in CH4 and C2H6 compared to Wufeng-Longmaxi shale.

Advances in chromatography in the study of drug-plasma protein interactions

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α1 -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。

Exploration of fiber-based cation exchange adsorbents for the removal of monoclonal antibody aggregates

Cation exchange chromatography (CEX) is a widely used technique for the removal of monoclonal antibody (mAb) aggregates. At present, resins are mainly used for this purpose, as convective types of adsorbents such as membrane adsorbers (MAs) have often not demonstrated overall comparable performance for this particular application. Fiber-based adsorbents can overcome the current limitations of MAs with respect to permeability, binding capacity, and adsorbent cost, and could therefore be a viable alternative to resins for the removal of mAb aggregates. It has not been evaluated, however, whether and under which conditions the use of such adsorbents is feasible for this purpose.In the present study, the use of fiber-based CEX adsorbents for mAb aggregate removal was examined. Two types of fiber-based adsorbents, an uncontrolled grafted and a controlled grafted fiber-based adsorbent, were evaluated with respect to permeability, dynamic mAb binding capacity (DBC), resolution capabilities, and the performance in bind and elute (B/E) and frontal chromatography mode with respect to typical performance indicators. The permeabilities of the fiber-based adsorbents ranged from 200 to 1700 mD, making it possible to use the fiber-based adsorbents at larger bed heights than membrane adsorbers with fast mobile phase velocities. Antibody DBCs ranged from 20 to 41 g/L at 150 cm/h, and at higher mobile phase velocities exceeded the DBC of an existing resin material, Poros 50 HS, which has frequently been used for aggregate removal. Both fiber types showed good resolution capabilities of monomer and aggregates, and provided better resolution per column length than Poros 50 HS. Typical purity and yield constraints were fulfilled for both fiber types in both B/E and frontal chromatography mode for mobile phase velocities ranging up to 480 cm/h and 1060 cm/h. The overall performance of the controlled grafted fibers was found to be superior to the performance of uncontrolled grafted fiber-based adsorbents due to higher productivity and lower buffer consumption. The overall performance of the fiber-based adsorbents was found to be comparable to the performance of Poros 50 HS at typical operating conditions.The results in this study indicate that the use of fiber-based adsorbents for mAb aggregate removal is feasible with a performance that is comparable to the performance of an existing resin material. Depending on the cost of the adsorbents and the use scenario, the usage of such adsorbents could be beneficial.

Facile synthesis of bifunctional polymer monolithic column for tunable and specific capture of glycoproteins and phosphoproteins

Efficiently tunable capture of the glycosylated/phosphorylated proteins is critical to meet the need of in-depth glycoproteome and phosphoproteome studies. Reported here is a new bifunctional polymer monolithic column by introducing benzeneboronic acid and phosphonic acid onto monolithic column (denoted as poly (EDMA-co-VPBA-co-VPA) monolith) for tunable and specific enrichment of glycoproteins and phosphoproteins via switching different mobile phases. Based on boronate affinity and immobilized metal affinity, the as-prepared poly (EDMA-co-VPBA-co-VPA) monolith exhibited superior performance in selective separation of small molecules and biomacromolecules containing cis-diol/phosphate groups or not. And the frontal chromatography analysis showed that the binding capacity of the poly (EDMA-co-VPBA-co-VPA) monolith towards horseradish peroxidase (HRP, glycoprotein) or β-casein (phosphoprotein) is four-fold higher than that of bovine serum albumin (BSA, non-glycosylated/phosphorylated protein). Furthermore, combined with mass spectrometry identification, the successful application in specific enrichment of glycopeptides/phosphopeptides from tryptic digests of HRP/β-casein and direct capture of low abundant endogenous phosphopeptides from human serum proved great practicability in complex samples. This study provides a novel insight for fabricating the monolithic columns with multifunctionalization to facilitate further post-translational modification (PTM)-proteomics development.

Analysis and optimal design of batch and two-column continuous chromatographic frontal processes for monoclonal antibody purification.

The increasing demand for efficient and robust processes in the purification of monoclonal antibodies (mAbs) has recently brought frontal chromatography to the forefront. Applied during the polishing step, it enables the removal of high molecular weight aggregates from the target product, achieving high purities. Typically, this process is operated in batch using a single column, which makes it intrinsically subjected to a purity-yield tradeoff. This means that high purities can only be achieved at the cost of lowering the product yield and vice versa. Recently, a two-column continuous implementation of frontal chromatography, referred to as Flow2, was developed. Despite being able of alleviating the purity-yield tradeoff typical of batch operations, the increase in the number of process parameters complicates its optimal design, with the risk of not exploiting its full potential. In this study, we developed an ad hoc design procedure (DP) suitable for the optimization of both batch frontal chromatography and Flow2 in terms of purity, yield, and productivity. This procedure provided similar results as a multiobjective optimization based on genetic algorithm but with lower computational effort. Then, batch and Flow2 operated at their optimal conditions were compared. Besides showing a more favorable Pareto front of yield and productivity at a specified purity, the Flow2 process demonstrated improved robustness compared to the batch process with respect to modifications in the loading linear velocity, washing buffer ionic strength and loading time, thus providing an appealing operation for integrated processes.

One-minute highly selective Cr(VI) determination at ultra-trace levels: An ICP-MS method based on the on-line trapping of Cr(III)

An analytical method derived from the coupling of frontal chromatography (FC) with Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) is proposed for the fast determination of Cr(VI) ultra-traces. The insertion of a short, homemade column filled with a strong cationic exchange resin in the flow-path of a commercial ICP-MS allows on-line trapping of cationic Cr(III) and elution of anionic Cr(VI). As a result, only the Cr(VI) front reaches the detector. This separation mechanism enables the highly selective quantification of Cr(VI) ultra-traces (LOD = 0.026 µg/kg - defined as 3 s of 10 replicated measurements of a 0.050 µg/kg solution) over a wide linearity range (tested up to 1024 µg/kg), even in the presence of Cr(III) concentration as high as 50 mg/kg. Key advantages of the proposed method are the extremely short analysis time (one minute), together with the simplicity and cost-effectiveness of the modifications applied over a commercial ICP-MS instrumental configuration. No time- or chemical-consuming pretreatments are needed: it is only necessary to acidify the sample prior Cr(VI) determination, as normally performed for common ICP-MS analysis. The applicability of the method was demonstrated over mineral water samples and toy migration solutions.

Derivation of adsorption capacity and adsorption isotherm by a single adsorbate concentration in liquid–solid system

A novel approach was presented as a strategy for deriving adsorption capacity and isotherm by a single adsorbate concentration in the liquid–solid system with experimental proof utilizing frontal chromatography (FC). A breakthrough curve equation presented in this work shows the dependence of adsorbate concentration in feed solution on time in FC and can be used to derive three characteristic binding parameters n (the number of the adsorbates bound to adsorbents), K (the adsorption equilibrium constant of adsorbates) and na (the total number of the adsorption sites). Moreover, the adsorption capacity and isotherm could be derived by a single breakthrough curve, even part of it. The equation was verified with the breakthrough curves of some arene and proteins on reversed-phase, ion-exchange, hydrophobic and affinity media, showing good fitness and consistent with the classical static method and elution peak method. Besides, all the adsorption data were referred to the type I adsorption of the Langmuir isotherm. This work presented new insight that utilizing a single breakthrough curve to determine adsorption parameters and isotherms and may provide some perspective for the determination of physico-chemical property and performance study of very rare substances.

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.