Prediction of protein retention times in gradient hydrophobic interaction chromatographic systems

A two-step methodology has been developed for the prediction of protein retention time in linear-gradient HIC systems. Isocratic retention parameters were determined from ln(k')-salt concentration plots for a number of commercially available proteins with a range of properties. Quantitative structure property relationship (QSPR) models based on a support vector machine (SVM) approach were generated for predicting isocratic retention parameters for proteins not included in the model generation. The predicted parameters were then used to calculate protein gradient retention times and the results indicate that this approach is well suited for predicting experimental gradient retention data. The approach presented in this paper may have implications for HIC methods development at both the bench and process scales.     

Prediction of protein secondary structure content using support vector machine

In this paper, the support vector machine was trained to grasp the relationship between the pair-coupled amino acid composition and the content of protein secondary structural elements, including -helix, 3₁₀-helix, π-helix, β-strand, β-bridge, turn, bend and the rest random coil. Self-consistency and cross validation tests were made to assess the performance of our method. Results superior to or competitive with the popular theoretical and experimental methods have been obtained.     

Prediction of retention times of proteins in hydrophobic interaction chromatography using only their amino acid composition

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 (MSE(JK)) 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.     

Prediction of retention times and peak widths in temperature-programmed gas chromatography using the finite element method

Optimization of separations in gas chromatography is often a time-consuming task. However, computer simulations of chromatographic experiments may greatly reduce the time required. In this study, the finite element method was used to predict the retention times and peak widths of three analytes eluting from each of four columns during chromatographic separations with two temperature programs. The data acquired were displayed in predicted chromatograms that were then compared to experimentally acquired chromatograms. The differences between the predicted and measured retention times were typically less than 0.1%, although the experimental peak widths were typically 10% larger than expected from the idealized calculations. Input data for the retention and peak dispersion calculations were obtained from isothermal experiments, and converted to thermodynamic parameters.     

Mixed retention mechanism of proteins in weak anion-exchange chromatography

Using four commercial weak anion-exchange chromatography (WAX) columns and 11 kinds of different proteins, we experimentally examined the involvement of hydrophobic interaction chromatography (HIC) mechanism in protein retention on the WAX columns. The HIC mechanism was found to operate in all four WAX columns, and each of these columns had a better resolution in the HIC mode than in the corresponding WAX mode. Detailed analysis of the molecular interactions in a chromatographic system indicated that it is impossible to completely eliminate hydrophobic interactions from a WAX column. Based on these results, it may be possible to employ a single WAX column for protein separation by exploiting mixed modes (WAX and HIC) of retention. The stoichiometric displacement theory and two linear plots were used to show that mechanism of the mixed modes of retention in the system was a combination of two kinds of interactions, i.e., nonselective interactions in the HIC mode and selective interactions in the IEC mode. The obtained U-shaped elution curve of proteins could be distinguished into four different ranges of salt concentration, which also represent four retention regions.     

Mathematical correlations for predicting protein retention times in hydrophobic interaction chromatography

In our work we performed a combinatorial synthesis in aqueous medium to prepare peptide libraries from which we would select amino acid sequences with binding properties towards estrogens. We prepared an affinity solid-phase by using a tetrapeptide with good selectivity and affinity towards the estradiol (K> 10(4) M(-1)). Samples of estrogens in buffer, in tap water and in river water were applied to our column in which they were retained (k' > 116). These could only be eluted in a few millilitres of methanol mobile phase. In all cases there were quantitative recoveries. The pre-concentration studies were promising.     

Prediction of peptide retention times in reversed-phases high-performance liquid chromatography during linear gradient elution

The retention behavior of 100 peptides was studied during high-performance liquid chromatography on a C₁₈ column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in a linear gradient elution system. Retention times of the peptides were linearly related to the logarithm of the sum of Rekker's constants (R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977, p. 301) for the constituent amino acid. Assuming this relationship, the best fit constants for this system were computed by non-linear multiple regression analysis. Using the new constants, it is possible to predict retention times for a wide variety of peptides at any slope of linear gradient, if the amino acid composition is known. It also enables accurate prediction of the retention time of peptides, whose amino acid composition in not known, after an analytical run with an alternate gradient.     

Prediction of retention times for anions in linear gradient elution ion chromatography with hydroxide eluents using artificial neural networks

The feasibility of using an artificial neural network (ANN) to predict the retention times of anions when eluted from a Dionex AS11 column with linear hydroxide gradients of varying slope was investigated. The purpose of this study was to determine whether an ANN could be used as the basis of a computer-assisted optimisation method for the selection of optimal gradient conditions for anion separations. Using an ANN with a (1, 10, 19) architecture and a training set comprising retention data obtained with three gradient slopes (1.67, 2.50 and 4.00 mM/min) between starting and finishing conditions of 0.5 and 40.0 mM hydroxide, respectively, retention times for 19 analyte anions were predicted for four different gradient slopes. Predicted and experimental retention times for 133 data points agreed to within 0.08 min and percentage normalised differences between the predicted and experimental data averaged 0.29% with a standard deviation of 0.29%. ANNs appear to be a rapid and accurate method for predicting retention times in ion chromatography using linear hydroxide gradients.     

Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

Prediction of protein retention at gradient elution conditions in ion-exchange chromatography.

This work presents a prediction procedure for protein retention in ion-exchange chromatography, where two linear gradient experiments of different length give the protein retention time at other linear gradients. The procedure predicts the retention time of early and late eluting proteins with similar precision and predictions by extrapolation deviate approximately 3% or less from the experimental retention times. By using the ionic strength, this procedure predicts protein retention times obtained with divalent ions in the eluent more accurately than a well-established procedure that uses the protein co-ion concentration.     

Simultaneous chemiluminescence determination of promazine and fluphenazine using support vector regression

In this work, chemiluminescence (CL) behaviors of two selected phenothiazines, namely promazine and fluphenazine hydrochloride, were investigated for their simultaneous determination using oxidation of Ru(bipy)₃²⁺ by Ce⁴⁺ ions in acidic media. This method is based on the kinetic distinction of the CL reactions of fluphenazine and promazine with Ru(bipy)₃²⁺ and Ce⁴⁺ system in a sulfuric acid medium. Least square support vector regression models were constructed for relating concentrations of both compounds to their CL profiles. The parameters of the model consisting of σ² and γ were optimized using all possible combinations of σ² and γ to select the model with the minimum root mean square cross validation. Under optimized conditions, the univariate calibration curve was linear over the concentration ranges of 0.4-30.0 μg mL⁻¹ and 0.07-5.0 μg mL⁻¹ with detection limits of 0.1 μg mL⁻¹ and 0.04 μg mL⁻¹ for promazine and fluphenazine, respectively. The influence of potential interfering substances on the determination of promazine and fluphenazine were studied. The proposed method was used for simultaneous determination of both compounds in synthetic mixtures and in spiked human plasma.     

Stationary phase contributions to retention in high-performance anion-exchange protein chromatography: ligand density and mixed mode effects

Several anion-exchange stationary phases (based on polyethyleneimine-coated silica) were synthesized so as to vary in ligand density and hydrophobicity. These materials were first examined for hemoglobin-binding capacity and then evaluated chromatographically. Protein binding, retention and resolution increased concomitantly with ligand density. Ferritin (molecular weight 440,000) could not be eluted from the more highly-charged surfaces, but was desorbed from a low ligand density support. The above parameters also varied with the hydrophobic character of the stationary phase. Retention and resolution increased as more hydrophobic moieties were added. Data from a non-ionic hemoglobin-binding assay correlated reasonably well with anticipated matrix hydrophobicities. Possible explanations and applications of the observed phenomena are discussed.     

Prediction of the retention in thin layer chromatography screening systems by atomic contributions

A novel atomic-contribution system for predicting of R_M values is presented and validated on 13 thin layer chromatography screening systems on silica gel, where the large experimental datasets (198-761 R_M values) are available. The R_M is predicted with error less than 0.5 in majority of solutes (besides several outliers), which corresponds to difference in R_F equal to 0.28 in the worst case. The system was validated by dividing the data into training and validation datasets, proving its accuracy. The main reason of larger errors in outliers are: large conjugated heterocycles, quarternary ammonium cations, large amount of polar atoms or very simple but unique molecules. The calculations are very easy and can be performed on free software or even manually. The presented method can be used in the retention prediction of new solutes in existing chromatographic screening systems.     

Prediction of peptide retention times in normal-phase liquid chromatography with only a single gradient run.

Previous studies of peptide separation by normal-phase liquid chromatography have shown a linear relationship between the logarithm of the capacity factor and the logarithm of the volume fraction of modifier in the mobile phase. This permitted the use of a model to predict isocratic and gradient retention times based on data obtained by two initial gradient runs. In the present study, chromatographic behavior of 25 peptides in normal-phase liquid chromatography with isocratic elution have been studied and a linear relationship between the slope (S) and intercept [log k(0)] was obtained. This relationship was combined with the algorithm of prediction reported in the previous paper. The prediction of peptide retention times with only a single experimental gradient retention data was investigated.