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Background Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco's activity could have a significant impact on photosynthetic efficiency and crop yield.Results This paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity. Conclusions We demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco's structural and functional properties. [ABSTRACT FROM AUTHOR]

Highlights • HIC column with mobile phase containing no salt and pH < 3 • HIC column enables separation of protein by size and small molecules by hydrophobicity • Online coupling HIC-RP columns for analysis of polysorbate in protein formulation. • Online HIC-RP columns can separates mixtures unresolved by either HIC or RPLC Abstract Polysorbates are complex mixtures of over a thousand components with a wide range of hydrophobicity. This paper describes a methodology for characterization of heterogeneity and stability of polysorbates in therapeutic protein formulation. The method utilizes on-line coupling of a hydrophobic interaction chromatography (HIC) column with reverse phase liquid chromatography (RPLC) and charged aerosol detection (CAD)/mass spectrometer (MS). The addition of a low concentration of formic acid and organic solvent (e.g. 0.05% formic acid and 3% acetonitrile) in the mobile phase enables the use of the HIC column to separate small molecule excipients (including major components of polysorbates) and the large protein molecules by a mixed mechanism of size exclusion chromatography (SEC) and RPLC. The protein and the charged excipients, which elute early from the HIC column, are directed by a switching valve to waste. The polysorbates and other neutral excipients, which elute later from the HIC column, are directed to the RPLC column for further separation. The separated polysorbate components are detected by CAD, and characterized by MS. This method has been used to characterize the degradation of polysorbate 80 (PS80) in placebo and protein formulations. Our studies have revealed different degradation behavior of PS80 in placebo vs. protein formulation. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) constitute a rapidly growing biopharmaceutical sector. However, their growth is impeded by high failure rates originating from failed clinical trials and developability issues in process development. There is, therefore, a growing need for better in silico tools to aid in risk assessment of mAb candidates to promote early-stage screening of potentially problematic mAb candidates. In this study, a quantitative structure–activity relationship (QSAR) modelling workflow was designed for the prediction of hydrophobic interaction chromatography (HIC) retention times of mAbs. Three novel descriptor sets derived from primary sequence, homology modelling, and atomistic molecular dynamics (MD) simulations were developed and assessed to determine the necessary level of structural resolution needed to accurately capture the relationship between mAb structures and HIC retention times. The results showed that descriptors derived from 3D structures obtained after MD simulations were the most suitable for HIC retention time prediction with a R2 = 0.63 in an external test set. It was found that when using homology modelling, the resulting 3D structures became biased towards the used structural template. Performing an MD simulation therefore proved to be a necessary post-processing step for the mAb structures in order to relax the structures and allow them to attain a more natural conformation. Based on the results, the proposed workflow in this paper could therefore potentially contribute to aid in risk assessment of mAb candidates in early development. [ABSTRACT FROM AUTHOR]

Trypsin is a protease widely used in several industrial areas for leather and meat softening and to produce enzymatic detergents, among others applications. The high demand for this enzyme has motivated the development of purification, stabilization and immobilization methods Formation of insoluble complexes between proteins and polyelectrolytes is a methodology that may include these features. The aim of this paper is to give evidence for a novel methodology that combines precipitation of the insoluble trypsin-alginate complex and hydrophobic interaction chromatography. This methodology allows the interaction between trypsin and alginate and their separation when necessary. It could be applied to isolation, stabilization and/or immobilization of trypsin. Isothermal titration calorimetry experiments showed that 232 μmol of trypsin interacts electrostatically with 1 g of alginate to form an insoluble complex that can be separated from soluble contaminants by decantation. Dynamic light scattering experiments confirmed the calorimetric results and allowed measuring the R h of the soluble complex at pH 3.5 (185 nm). When the optimal conditions were applied to precipitate commercially available trypsin, the recovery of the precipitation was around 92%. Finally, hydrophobic interaction chromatography allowed separating alginate from trypsin in order to obtain a polymer-free enzyme. [ABSTRACT FROM AUTHOR]

Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3–10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. [ABSTRACT FROM AUTHOR]

In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized. [ABSTRACT FROM AUTHOR]

Butyl-acrylate (BA) containing branched poly-(N-isopropyl acrylamide) or PNIPAM copolymer was grafted on filter paper to prepare composite membrane for carrying out temperature-responsive hydrophobic interaction membrane chromatography (HIMC) of proteins. The copolymer demonstrated two-step phase transition behavior due to the presence of BA and PNIPAM, making the changeover from hydrophilic to hydrophobic state more tunable. The feasibility of using the temperature-responsive membrane was demonstrated by controlling human immunoglobulin G (hIgG) binding and release using temperature change. Higher temperature promoted protein binding and lower temperature favored protein release in this process, which was carried out using lyotropic salt-free mobile phase. The purification of monoclonal antibody (mAb) hIgG1-CD4 from simulated CHO cell culture supernatant was carried out to demonstrate practical application for the novel temperature-responsive membrane. [ABSTRACT FROM AUTHOR]

Aggregates of monoclonal antibody (mAb) therapeutics, due to their perceived impact on immunogenicity, are recognized as a critical quality attribute by the regulatory authorities as well as the industry. Hence, removal of aggregates is a key objective of bioprocessing. At present, this is achieved by a combination of two or more orthogonal chromatographic steps with possible modalities of ion exchange, hydrophobic interaction and mixed mode. A two-stage chromatographic purification process consisting of ion-exchange and hydrophobic interaction modes is proposed in this paper for effective and efficient control of aggregates for a mAb therapeutic. The proposed scheme does not require any intermediate processing of the process stream. Further, baseline separation is achieved for monomer and aggregates resulting in robust performance. This was possible because the proposed operational scheme allowed for an addition of selectivities of the two chromatography modes vs. the traditional two column scheme where part of the separation of aggregates achieved by the first column is lost upon pooling. The proposed process scheme yielded improved separation of aggregates (0% vs. 1–2%) at >95% recovery and reduced overall process time (6 h vs . 14 h) for a typical application. Further, clearance of host cell proteins was also shown to have improved with the suggested process scheme. Successful implementation of the proposed scheme has been demonstrated for two different monoclonal antibody therapeutic products. [ABSTRACT FROM AUTHOR]

Chromatography is a technique widely used in the purification of biopharmaceuticals, and generally consists of several chromatographic steps. In this work, Hydrophobic Interaction Chromatography (HIC) is investigated as a polishing step for the purification of therapeutic proteins. Adsorption mechanisms in hydrophobic interaction chromatography are still not completely clear and a limited amount of published data is available. In addition to new data on adsorption isotherms for some proteins (obtained both by high-throughput and frontal analysis method), and a comparison of different models proposed in the literature, two different approaches are compared in this work to investigate HIC. The predictive approach exploits an in-house code that simulates the behavior of the component in the column using the model parameters found from the fitting of experimental data. The estimation approach, on the other hand, exploits commercial software in which the model parameters are found by the fitting of a few experimental chromatograms. The two approaches are validated on some bind-elute runs: the predictive approach is very informative, but the experimental effort needed is high; the estimation approach is more effective, but the knowledge gained is lower. The second approach is also applied to an in-development industrial purification process and successfully resulted in predicting the behavior of the system, allowing for optimization with a reduction in the time and amount of sample needed. [ABSTRACT FROM AUTHOR]

Abstract: Phycocyanin – a major phycobiliprotein constitutively produced by many cyanobacteria hold several promising applications in diagnostics, biomedical research, and therapeutics. This paper discusses a novel rapid method for the purification of cyanobacterial phycocyanin (C-PC) from Phormidium fragile using hydrophobic interaction chromatography. The protein was extracted and concentrated by grinding under liquid nitrogen and ammonium sulfate fractionation. C-PC was purified by single step hydrophobic interaction chromatography. Purified phycocyanin showed absorbance maximum (λ max) at 624nm. The criterion of purity (R) achieved was 4.52. Phycocyanin to phycoerythrin and phycocyanin to allophycocyanin purity ratio were 3.85 and 7.49, respectively. The purified protein showed a pI of 5.2 and has two subunits with molecular mass of 19 and 20kDa each, corresponding to its highly reported α and β subunits. The subunits of phycocyanin were confirmed by their bilin fluorescence using zinc assisted florescence enhancement technique. Intact C-PC was of 125kDa as determined by HPLC, suggested the (αβ)3 subunit assembly. Results obtained by this method in terms of purity, recovery, process time, simplicity, and efficacy are much better than previous methodologies. Purified phycocyanin was further scrutinized for its antioxidant capacity and judged against five non-enzymatic antioxidants by FRAP assay. [Copyright &y& Elsevier]

Abstract: The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD600 =40) culture of recombinant E. coli, and an SK protein concentration of 1080mgl−1 was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18×105 in cell lysate to 0.53×105 IUmg−1 resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96×105 IUmg−1) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps. [Copyright &y& Elsevier]

Abstract: Protein retention in hydrophobic interaction chromatography is determined by protein physicochemical properties and by system characteristics. In this paper we present an attempt to determine the optimal operation conditions that would allow the separation of binary protein mixtures. The statistically significant system variables were determined, and then empirical models were obtained which explained more than 92% of variability in dimensionless retention time based on salt properties, ionic strength of the initial eluent and substitution degree of the resin. These variables were optimized in order to achieve the maximum retention time difference between two proteins in a mixture. The optimum operation conditions as predicted by the models were tested experimentally, showing a good agreement with predicted separation. We concluded that it would be possible to determine the system conditions that allow the maximum separation of two proteins based on the main system properties. The methodology proposed here presents potential to be applied to partially characterized systems, however, it could be improved if protein''s properties were included explicitly in the models. [Copyright &y& Elsevier]

Abstract: This paper gives a summary of different aspects for predicting protein behaviour in hydrophobic interaction chromatography (HIC). First, a brief description of HIC, hydrophobic interactions, amino acid and protein hydrophobicity is presented. After that, several factors affecting protein chromatographic behaviour in HIC are described. Finally, different approaches for predicting protein retention time in HIC are shown. Using all this information, it could be possible to carry out computational experiments by varying the different operating conditions for the purification of a target protein; and then selecting the best conditions in silico and designing a rational protein purification process involving an HIC step. [Copyright &y& Elsevier]

Abstract: This paper focuses on the prediction of the dimensionless retention time (DRT) of proteins in hydrophobic interaction chromatography (HIC) by means of mathematical models based on the statistical description of the amino acid surface distribution. Previous models characterises the protein surface as a whole. However, most of the time it is not the whole protein but some of its specific regions that interact with the environment. It seems much more natural to use local measurements of the characteristics of the surface. Therefore, the statistical characterisation of the distribution of an amino acid property on the protein surface was carried out from the systematic calculation of the local average of this property in a neighbourhood placed sequentially on each of the amino acids on the protein surface. This process allowed us to characterise the distribution of this property quantitatively using three main statistics: average, standard deviation and maximum. In particular, if the property considered is a hydrophobicity scale, these statistics allowed us to characterise the average hydrophobicity and the hydrophobic content of the most hydrophobic cluster or hotspot, as well as the heterogeneity of the hydrophobicity distribution on the protein surface. We tested the performance of the DRT predictive models based on these statistics on a set of 15 proteins. We obtained better predictive results with respect to the models previously reported. The best predictive model was a linear model based on the maximum. This statistic was calculated using an index of the mobilities of amino acids in chromatography. The predictive performance of this model (measured as the Jack Knife MSE) was 26.9% better than those obtained by the best model which does not consider the amino acid distribution and 19.5% better than the model based on the hydrophobic imbalance (HI). In addition, the best performance was obtained by a linear multivariable model based on the HI and the maximum. The difference between the experimental data and the prediction carried out by this model was smaller than those observed previously. In fact, this model obtained better predictive capacities than a previous linear multivariable model decreasing the Jack Knife MSE in 8.7%. In addition, this model allowed us to diminish the number of variables required, increasing, in this way, the degrees of freedom of the model. [Copyright &y& Elsevier]

Abstract: This paper focuses on the prediction of the dimensionless retention time of proteins (DRT) in hydrophobic interaction chromatography (HIC) by means of mathematical models based on characteristics of the surface hydrophobicity distribution. We introduce a new parameter, called hydrophobic imbalance (HI), obtained from the three-dimensional structure of proteins. This parameter quantifies the displacement of the superficial geometric centre of the protein when the effect of the hydrophobicity of each amino acid is considered. This parameter is simpler and less expensive than those reported previously. We use HI as a way to incorporate information about the surface hydrophobicity distribution in order to improve the prediction of DRT. We tested the performance of our DRT predictive models in a set of 15 proteins. This set includes four proteins whose DRTs are known as very difficult to predict. By means of the variable HI, it was possible to improve the predictive characteristics obtained by models based on the average surface hydrophobicity (ASH) by 9.1%. Also, we studied linear multivariable models based on characteristics determined from the HI. By using this multivariable model, a correlation coefficient of 0.899 was obtained. With this model, we managed to improve the predictive characteristics shown by previous models based on ASH by 31.8%. [Copyright &y& Elsevier]

Abstract: This paper focuses on the prediction of the dimensionless retention time of proteins (DRT) in hydrophobic interaction chromatography (HIC) by means of mathematical models based, essentially, only on aminoacidic composition. The results show that such prediction is indeed possible. Our main contribution was the design of models that predict the DRT using the minimal information concerning a protein: its aminoacidic composition. The performance is similar to that observed in models that use much more sophisticated information such as the three-dimensional structure of proteins. Three models that, in addition to the amino acid composition, use different assumptions about the amino acids tendency to be exposed to the solvent, were evaluated in 12 proteins with known experimental DRT. In all the cases analyzed, the model that obtained the best results was the one based on a linear estimation of the aminoacidic surface composition. The models were adjusted using a collection of 74 vectors of aminoacidic properties plus a set of 6388 vectors derived from these using two mathematical tools: k-means and self-organizing maps (SOM) algorithms. The best vector was generated by the SOM algorithm and was interpreted as a hydrophobicity scale based partly on the tendency of the amino acids to be hidden in proteins. The prediction error (MSEJK) obtained by this model was almost 35% smaller than that obtained by the model that supposes that all the amino acids are completely exposed and 40% smaller than that obtained by the model that uses a simple correction factor considering the general tendency of each amino acid to be exposed to the solvent. In fact, the performance of the best model based on the aminoacidic composition was 5% better than that observed in the model based on the three-dimensional structure of proteins. [Copyright &y& Elsevier]

Trichoderma reesei cellulase complex was fractionated using hydrophobic interaction chromatography with a phenyl-Sepharose column. Using a linear gradient of ammonium sulphate in the eluent buffer, a selective separation of endoglucanases was obtained at 15 °C with a four-fold increase in specific activity. [ABSTRACT FROM AUTHOR]

A preferential interaction quadratic isotherm model for hydrophobic interaction chromatographic systems is presented in this paper. In this isotherm, the nonlinear effect of salt on the capacity factor is described using the preferential interaction model developed by Perkins et al. [J. Chromatogr. A, 766 (1997) 1]. This is then coupled with a quadratic nonlinear isotherm to describe nonlinear adsorption behavior at high solute concentrations. The resulting preferential interaction quadratic isotherm is examined for its ability to describe solute adsorption behavior under both linear and nonlinear conditions over a wide range of salt concentrations in HIC systems. The results indicate that this isotherm is well suited for predicting nonlinear adsorption behavior in HIC systems for both proteins and low-molecular mass HIC displacers. [Copyright &y& Elsevier]

Exosomes are membrane‐secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary‐channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5–30 cm) and loading flow rates (0.2–0.5 ml/min). A loading capacity of 5.4 × 1012 exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications. [ABSTRACT FROM AUTHOR]

Purpose: The study of interactions between humic substances (HSs) and soil filamentous fungi is the key to understanding the sustainable soil functioning. The present work aims to examine the decomposition of HSs by filamentous dark-pigmented fungus Alternaria alternatа under the laboratory conditions and to determine the effect of easily assimilable organic carbon on this process. Analyzing such polydisperse substances like HSs by a complex integrated methodology makes it possible to explore the data on their decomposition by microorganisms. Materials and methods: To achieve the aforementioned goals, we used chromatographic and spectroscopic approaches: low-pressure size-exclusion and hydrophobic interaction chromatography accompanied by absorption and fluorescence spectroscopy. To determine the effect cometabolism conditions produced on HS decomposition, two types of carbon substrates were added to the nutrient media: easily assimilable organic carbon (standard 0.3% or reduced 0.03% sucrose content) and hardly assimilable organic carbon (HSs), as well as their combinations. Five HS samples of different organic matter origin have been inspected: potassium humates (HPs) and humic acids (HAs) from coal, peat, and lignosulfonate. Correlation matrix and principal component analysis (PCA) were calculated for comprehensive data analysis. Results and discussion: Transformations of the investigated HSs under fungal cultivation lead to the increase in the low molecular weight fraction, rise of hydrophilic fraction, enlargement of absorbance ratio A250/A365, shortening of the emission wavelength of the humic-type fluorescence, and growth in the fluorescence quantum yield measured with excitation at 355 nm. A positive correlation was observed between the accumulation of fungal biomass and the degree of HS decomposition. PCA analysis confirms that the difference in the results of HS decomposition largely depends on the sucrose content and the nature of HSs. We divided all the HS samples into four groups according to the degree of HS decomposition: original HS solutions, HPs altered using fungal cultivation at 0.03% sucrose, HAs after fungal cultivation at 0.03% sucrose, and finally, HSs (both HPs and HAs) after fungal cultivation at 0.3% sucrose. Conclusions: In the laboratory experiments, we showed that (1) the isolated HAs were more effectively degraded than the parent HPs, and this process was more pronounced at a reduced sucrose content, and (2) the decomposition of stable organic compounds (HSs) was activated by the easily assimilable carbon sources (especially 0.3% sucrose) being present. We assume that it is the easily assimilable organic carbon that most likely triggers the HS degradation working as the priming effect in natural environments. [ABSTRACT FROM AUTHOR]

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) constitute a rapidly growing biopharmaceutical sector. However, their growth is impeded by high failure rates originating from failed clinical trials and developability issues in process development. There is, therefore, a growing need for better in silico tools to aid in risk assessment of mAb candidates to promote early-stage screening of potentially problematic mAb candidates. In this study, a quantitative structure–activity relationship (QSAR) modelling workflow was designed for the prediction of hydrophobic interaction chromatography (HIC) retention times of mAbs. Three novel descriptor sets derived from primary sequence, homology modelling, and atomistic molecular dynamics (MD) simulations were developed and assessed to determine the necessary level of structural resolution needed to accurately capture the relationship between mAb structures and HIC retention times. The results showed that descriptors derived from 3D structures obtained after MD simulations were the most suitable for HIC retention time prediction with a R2 = 0.63 in an external test set. It was found that when using homology modelling, the resulting 3D structures became biased towards the used structural template. Performing an MD simulation therefore proved to be a necessary post-processing step for the mAb structures in order to relax the structures and allow them to attain a more natural conformation. Based on the results, the proposed workflow in this paper could therefore potentially contribute to aid in risk assessment of mAb candidates in early development.

Hydrophobic interaction chromatography (HIC) is often used for purifying proteins. The prediction of the adsorption equilibria in such systems is a difficult task as they depend on the pH value and the influence of the salts, which are added for controlling the solvent properties. This is especially tedious when salt mixtures are used, which can have positive or negative cooperative effects on the adsorption. A mathematical model to describe these complex effects of the salts was recently introduced by our group. In that model, the influence of the salts is described by a Taylor series expansion in the individual ion molarities. However, in our previous work, only the loading of the adsorbent at a given constant protein concentration in the liquid phase cp = 0.01 mM was considered and correlated as a function of the salt composition at constant ionic strength. In the present study, that model is extended to other protein concentrations and it is shown that the Taylor series expansion works well for a wide range of cp. The model parameters are found to depend on cp, but only in a simple way. The dependence of the model parameters on cp was correlated using a new three parameter equation. From that correlation, the loading qp can be determined for any value of cp. Hence, the entire adsorption isotherm is known for any salt composition. The approach is illustrated using experimental data from previous studies of our group on the adsorption of bovine serum albumin, lysozyme, polyethylene glycol, and di‐PEGylated lysozyme on the mildly hydrophobic resin Toyopearl PPG‐600 M. The pH values are between 4.0 and 7.0. Binary and ternary mixtures of ammonium chloride, sodium chloride, ammonium sulfate, and sodium sulfate as well as the single salts are used at overall ionic strengths between 1,500 and 4,200 mM. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 [ABSTRACT FROM AUTHOR]

Penicillin acylase (PA, EC 3.5.1.11) is used as a raw material in the production of semi-synthetic penicillins. Although there are many methods for PA purification, affinity chromatography is advantageous as it provides efficient one step purification. In this study, poly(2-hydroxyethyl methacrylate) based cryogel column containing hydrophobic N-methacryloyl-L-tryptophan (MATrp) functional monomer as a ligand was prepared. Interaction of MATrp with amino acids in PA structure is the basis of hydrophobic interaction chromatography in this study. PHEMA and PHEMATrp cryogel columns were characterized by surface area measurements, infrared spectroscopy, swelling tests, elemental analysis and scanning electron microscopy (SEM). Initial PA concentration, pH, effect of temperature, amount of ligand, flow rate, ionic strength and time on PA adsorption on PHEMATrp cryogel were investigated. Optimum pH was determined as 5.0 for PA adsorption and maximum adsorption capacity was obtained as 6.40 mg/g. It was observed that adsorption capacity increased with the increasing of temperature. Also, PA adsorption increased up to 0.25 M salt concentration and decreased in higher salt concentrations. Data obtained in this affinity system suggests that hydrophobic interactions are dominant. In the last stage of the study, PA was purified fromPenicillium chrysogenumwith 76.3% yield and 332.3 purification factor. [ABSTRACT FROM AUTHOR]

Lipases from Geotrichum candidum were produced in two different medium: A = 12 % (w/v) clarified corn steep liquor (CCSL) + 0.6 % (w/v) soybean oil (SO) and B = 3.5 % (w/v) yeast hydrolysate (YH) + 0.7 % (w/v) SO. Lipases were partially purified from both media by hydrophobic interaction chromatography using 3.0 mol L of NaCl as mobile phase, and they were characterized in the crude and partially purified forms. The recovery of lipase activity from CCSL and YH via HIC were 96 and 94.3 %, and the purification factors were 44.3 and 86.7-fold, respectively. All evaluated lipases had similar optimum pH (7.0-7.7), but, for the CCSL crude lipase, optimum temperature (47 °C) was 10 °C higher than others lipases evaluated. CCSL crude lipase possessed a higher thermo stability than YH crude lipase, e.g., at 37 °C (pH 7.0) the half-life of CCSL crude lipase was 19.25 h and at pH 8.0 (30 °C) the half-life was 48 h, which are five and ten times higher than with YH crude lipase, respectively. On the other hand, the YH crude lipase possessed a higher catalytic constant ( k = 2.3 min) but with almost the same catalytic efficiency ( K / k = 32.12 mg mL min) in relation to CCSL crude lipase. The lipases differ in biocatalytic properties between substrates, suggesting that the two lipases can be employed for different applications. [ABSTRACT FROM AUTHOR]

Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in downstream processing of biopharmaceuticals. During HIC, salts are the governing additives contributing to binding strength, binding capacity, and protein solubility in the liquid phase. A relatively recent approach to increase the dynamic binding capacity (DBC) of HIC adsorbers is the use of salt mixtures. By mixing chaotropic with kosmotropic salts, the DBC can strongly be influenced. For salt mixtures with a higher proportion of chaotropic than kosmotropic salt, higher DBCs were achieved compared with single salt approaches. By measuring the surface tensions of the protein salt solutions, the cavity theory-proposed by Melander and Horváth-that higher surface tensions lead to higher DBCs, was found to be invalid for salt mixtures. Aggregation temperatures of lysozyme in the salt mixtures, as a degree of hydrophobic forces, were correlated to the DBCs. Measuring the aggregation temperatures has proven to be a fast analytical methodology to estimate the hydrophobic interactions and thus can be used as a measure for an increase or decrease in the DBCs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:346-354, 2016 [ABSTRACT FROM AUTHOR]

β-Glucosidase (β- d-glucoside glucohydrolase, EC 3.2.1.21) is a catalytic enzyme present in both prokaryotes and eukaryotes that selectively catalyzes either the linkage between two glycone residues or between glycone and aryl or alkyl aglycone residue. Growing edible mushrooms in the soil with increased cellulose content can lead to the production of glucose, which is a process dependent on β-glucosidase. In this study, β-glucosidase was isolated from Agaricus bisporus (white button mushroom) using ammonium sulfate precipitation and hydrophobic interaction chromatography, giving 10.12-fold purification. Biochemical properties of the enzyme were investigated and complete characterization was performed. The enzyme is a dimer with two subunits of approximately 46 and 62 kDa. Optimum pH for the enzyme is 4.0, while the optimum temperature is 55 °C. The enzyme was found to be exceptionally thermostable. The most suitable commercial substrate for this enzyme is p-NPGlu with K and V values of 1.751 mM and 833 U/mg, respectively. Enzyme was inhibited in a competitive manner by both glucose and δ-gluconolactone with IC values of 19.185 and 0.39 mM, respectively and K values of 9.402 mM and 7.2 µM, respectively. Heavy metal ions that were found to inhibit β-glucosidase activity are I, Zn, Fe, Ag, and Cu. This is the first study giving complete biochemical characterization of A. bisporus β-glucosidase. [ABSTRACT FROM AUTHOR]

This paper describes a method for the plasmid DNA purification, which includes an ammonium sulphate precipitation, followed by hydrophobic interaction chromatography (HIC) using Phenyl Sepharose 6 Fast Flow (low sub). The use of HIC took advantage of the more hydrophobic character of single stranded nucleic acid impurities as compared with double-stranded plasmid DNA.O DNA plasmidial tem sua aplicação na terapia gênica, tendo que apresentar elevado grau de pureza. Um método de purificação foi descrito neste trabalho, que inclui precipitação com sulfato de amônio seguida da cromatografia de interação hidrofóbica, usando a resina phenyl sepharose 6 fast flow. Ocorreu aumento da pureza das amostras analisadas, em torno de 30% após a precipitação com sulfato de amônio. Houve significativa redução dos contaminantes após a eluição das amostras na cromatografia de interação. O maior rendimento obtido foi 53% e fator de purificação 3,1 vezes após a eluição na cromatografia de interação hidrofóbica. A vantagem deste método é a possibilidade de explorar o caráter hidrofóbico, constituído de material genético de fita simples, tais como RNA e DNA genômico e proteínas, que podem interferir na liberação deste material para possíveis aplicações em terapia gênica.

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A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol–ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96 % can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2 %, 92.4 %, and 2.8 × 107 IU/mg, respectively, in IEC mode, and 83.4 %, 95.2 %, and 4.3 × 107 IU/mg, respectively, in HIC mode. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes. [ABSTRACT FROM AUTHOR]

Background: Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results: Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions: Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes. [ABSTRACT FROM AUTHOR]

□ In this study, an efficient purification system was developed using a packed-bed column for the purification of recombinant green fluorescent protein (GFP) from clarified feedstock. A series of hydrophobic ligands [phenyl (low sub), phenyl (high sub), butyl, and octyl] was screened at bench scale and the optimum concentration of salt in binding buffer was determined by precipitating the sample with (NH4)2SO4. The optimum salt concentration used in binding buffer is 30%(w/v) (NH4)2SO4in 20 mM sodium phosphate buffer at pH 8.0. Phenyl (high sub) ligand showed the best result of GFP purification in terms of yield and purity. Maximum recovery (82%) of recombinant GFP was achieved with phenyl (high sub) ligand. The optimum elution buffer for scaled-up process was determined by conducting a stepwise elution with 0–15%(w/v) (NH4)2SO4salt solution and the result showed that the GFP absorbed at the ligand was mostly eluted by using a buffer with the absence of (NH4)2SO4. The effect of GFP concentration in initial sample on the dynamic binding capacity was also studied by running a scaled-up hydrophobic interaction chromatography using HiPrep™ HIC Phenyl (high sub) 16/10 20mL packed-bed column connected to Äkta FPLC. [ABSTRACT FROM AUTHOR]

ABSTRACT A novel dual-retention mechanism mixed-mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high-salt-concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low-salt-concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion-exchange chromatography and HIC columns. The results indicated that the novel dual-retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a '2D column'. In addition, the mixed retention mechanism of proteins on this '2D column' was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual-retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this '2D column', a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

It has been shown that the distribution features of the hydrophilic and hydrophobic fractions of humic substances (HSs) in the upper genetic horizons of undisturbed podzolic soils and litho-barrier podzols are similar. The concentrations of the separate HS fractions in the mineral horizons are largely determined by the soil mineralogy. In forest litters of secondary phytocenoses developed after the cutting of spruce forests, an increase in the content of hydrophilic compounds has been observed on loamy deposits at a decrease in the portion of lignin-like organic compounds. An increase in the portion of Al-Fe-humus compounds has been revealed in the mineral soil horizons of secondary phytocenoses. It has been proposed to use the degree of hydrophilicity ( D) for characterizing the features of the organic matter in taiga soils. An increase in the content of the fractions of occluded organic matter and that bound to minerals (density of 1.6-2.2 g/cm) has been observed in the eluvial horizons of a young cutover area. [ABSTRACT FROM AUTHOR]

ABSTRACT As alternative hydrophobic adsorbent for DNA adsorption, supermacroporous cryogel disks were synthesized via free radical polymerization. In this study, we have prepared two kinds of cryogel disks: (i) poly(2-hydroxyethyl methacrylate- N-methacryloyl- l-tryptophan) [p(HEMA-MATrp)] cryogel containing specific hydrophobic ligand MATrp; and (ii) monosize p(HEMA-MATrp) particles synthesized via suspension polymerization embedded into p(HEMA) cryogel structure to obtain p(HEMA-MATrp)/p(HEMA) composite cryogel disks. These cryogel disks containing hydrophobic functional group were characterized via swelling studies, Fourier transform infrared spectroscopy, elemental analysis, surface area measurements and scanning electron microscopy. DNA adsorption onto both p(HEMA-MATrp) cryogel and p(HEMA-MATrp)/p(HEMA) composite cryogels was investigated. Maximum adsorption of DNA on p(HEMA-MATrp) cryogel was found to be 15 mg/g polymer. Otherwise, p(HEMA-MATrp)/p(HEMA) composite cryogels significantly increased the DNA adsorption capacity to 38 mg/g polymer. Composite cryogels could be used repeatedly without significant loss on adsorption capacity after 10 repetitive adsorption-desorption cycles. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Nylon-6 capillary-channeled polymer (C-CP) fibers are used as the stationary phase for the hydrophobic interaction chromatography (HIC) separation of a synthetic protein mixture composed of ribonuclease A, lysozyme, and holotransferrin. Nylon is a useful polymer phase for HIC as it has an alkyl backbone, while the amide functionality is hydrophilic (in fact ionic) in nature. The combination of a nonporous polymer surface of the fiber phases and high column permeability yields very efficient mass transfer characteristics, as exhibited by narrowing of peak widths with increases in mobile phase linear velocity. Retention factors and resolution were evaluated at flow rates ranging from 0.5 to 9 mL/min (linear velocities of ca. 2 to 15 mm/s) and at gradient slopes between 3.3 and 20 %B/min. Optimum resolution was achieved by employing fast flow rates (9 mL/min) and slow gradients (3 %B/min), also resulting in the highest peak capacities. [ABSTRACT FROM AUTHOR]

R-phycoerythrin (R-PE) was purified from leafy gametophyte of Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) by a simple, scaleable procedure. Initially, phycobiliproteins were extracted by repeated freeze-thaw cycles, resulting in release from the algal cells by osmotic shock. Next, R-PE was recovered by applying the crude extract with a high concentration of (NH4)2SO4 salt directly to the expanded-bed columns loaded with phenyl-sepharose. An expanded-bed volume twice the settled-bed volume was maintained; then low (NH4)2SO4 concentration was used to develop the column. After two rounds of hydrophobic interaction chromatography (HIC), R-PE was purified by anion-exchange column. The method was also successful with free-living conchocelis of P. haitanensis. The purified R-PE was identified with electrophoresis, and absorption and fluorescence emission spectroscopy. The results were in agreement with those previously reported. The yield with a spectroscopic purity (OD565/OD280) higher than 3.2 (the ratio of A565/ A620 ≤ 0.02) was 1.4 mg · g−1 of leafy gametophyte of P. haitanensis. For the free-living conchocelis of P. haitanensis extract, R-PE could be purified successfully with only one round of HIC. The yield with a spectroscopic purity (OD565/OD280) higher than 3.2 (the ratio of A565/ A620 ≤ 0.02) was 5.0 mg · g−1 of free-living conchocelis of P. haitanensis. The method described here is a scaleable technology that allows a large quantity of R-PE to be recovered from the unclarified P. haitanensis crude extract. It is also a high protein recovery technology, reducing both processing costs and times, which enhances the value of this endemic Porphyra of China. [ABSTRACT FROM AUTHOR]

Interaction between proteins and stationary phase in hydrophobic interaction chromatography (HIC) is differentiated into two thermodynamic processes involving direct nonbonding/conformation interaction and surface hydrophobic effect of proteins, hence quantitatively giving rise to a binary linear relation between HIC retention time (RT) at concentrated salting liquid and ligand-protein binding free energy. Then, possible binding manners for 27 proteins of known crystal structures with hydrophobic ligands are simulated and analyzed via ICM flexible molecular docking and genetic algorithm, with results greatly consistent with experimental values. By investigation, it is confirmed local hydrophobic effects of proteins and nonbinding/conformation interaction between ligand and protein both notably influence HIC chromatogram retention behaviors, mainly focusing on exposed portions on the protein surface. [ABSTRACT FROM AUTHOR]

A new method for the direct detection of trypsin, chymotrypsin A and kallikrein from porcine pancreas by capillary zone electrophoresis is proposed. Optimum conditions for quantitative determination were investigated which also allow the unequivocal distinction of trypsin and chymotrypsin according to porcine or bovine source. For each enzyme good linearity and precision were obtained, with a detection limit of 5.0 μg mL−1. In addition, the formation of the complexes between trypsin and basic pancreatic trypsin inhibitor, as well as that between kallikrein and the same inhibitor, have been investigated. Such complexes can be clearly distinguished either from the unreacted enzyme or the excess inhibitor and their use for analytical purposes has been evidenced. The method is rapid and sensitive and has been applied to determine enzymes in several porcine pancreas extracts. The levels of the active compounds in various kinds of commercial preparations were easily determined with relative standard deviations ranging between 3.3 and 4.9%, and recoveries from 95.8% to 102.4%. [ABSTRACT FROM AUTHOR]

The authors have developed a rapid and convenient method for purification of a low molecular weight form (Δ10) of the bacterial plasminogen activator, staphylokinase. Recombinant staphylokinase is expressed in Escherichia coli, with an amino terminal extension that facilitated purification by immobilized metal-affinity chromatography. Purified staphylokinase is treated with human plasminogen, and the resulting truncated form is purified using a combination of immobilized metal affinity chromatography and hydrophobic interaction chromatography. Purified protein is characterized by amino terminal sequencing and in vitro plasminogen activation assay. [ABSTRACT FROM AUTHOR]