Estimation of Nonlinear Adsorption Isotherms in Gradient Elution RP-LC of Peptides in the Presence of an Adsorbing Additive.

ABSTRACT: In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

Combining Chemometric Models with Adsorption Isotherm Measurements to Study Omeprazole in RP-LC.

The adsorption of the proton-pump inhibitor omeprazole was investigated using RP-LC with chemometric models combined with adsorption isotherm modelling to study the effect of pH and type of organic modifier (i.e., acetonitrile or methanol). The chemometric approach revealed that omeprazole was tailing with methanol and fronting with acetonitrile along with increased fronting at higher pH. The increased fronting with higher pH for acetonitrile was explored using a pH-dependent adsorption isotherm model that was determined using the inverse method and it agreed well with the experimental data. The model indicated that the peaks exhibit more fronting at high pH due to a larger fraction of charged omeprazole molecules. This model could accurately predict the shape of elution profiles at arbitrary pH levels in the studied interval. Using a two-layer adsorption isotherm model, the difference between acetonitrile and methanol was studied at the lowest pH at which almost all omeprazole molecules are neutral. Omeprazole had adsorbate-adsorbate interactions that were similar in strength for the acetonitrile and methanol mobile phases, while the solute-adsorbent interactions were almost twice as strong with methanol. The difference in the relative strengths of these two interactions likely explains the different peak asymmetries (i.e., tailing/fronting) in methanol and acetonitrile. In conclusion, thermodynamic modelling can complement chemometric modeling in HPLC method development and increase the understanding of the separation.

Choice of Model for Estimation of Adsorption Isotherm Parameters in Gradient Elution Preparative Liquid Chromatography.

The inverse method is a numerical method for fast estimation of adsorption isotherm parameters directly from a few overloaded elution profiles and it was recently extended to adsorption isotherm acquisition in gradient elution conditions. However, the inverse method in gradient elution is cumbersome due to the complex adsorption isotherm models found in gradient elution. In this case, physicochemically correct adsorption models have very long calculation times. The aim of this study is to investigate the possibility of using a less complex adsorption isotherm model, with fewer adjustable parameters, but with preserved/acceptable predictive abilities. We found that equal or better agreement between experimental and predicted elution profiles could be achieved with less complex models. By being able to select a model with fewer adjustable parameters, the calculation times can be reduced by at least a factor of 10.

Characterization of antibody surface properties and selection of a diverse subset for development of a generic size-exclusion chromatography method.

Aggregates are an important quality attribute for biotherapeutics because they can affect the safety and efficacy of the drug and they are therefore routinely monitored, mainly with size-exclusion chromatography (SEC). However, there is often a need to tailor a SEC method to a specific molecule. This study describes the development of a generic SEC method tested on 138 antibodies with the variable domains taken from clinical stage antibodies. We report on the discovery of a subset of 12 antibodies that represents the full range of physiochemical properties found in the 138 antibodies. This subset is shown to be an efficient and reliable test set when developing chromatographic methods for antibodies. An understanding of the nature of the analyte-stationary phase interactions was gained when using this set with its wide range of physiochemical properties. Highly hydrophobic antibodies interact strongly with some modern silica hybrid materials causing the elution time to increase significantly, while a hydrophilically modified hybrid surface showed highly reduced interactions for the hydrophobic antibodies. Highly hydrophilic antibodies, on the other hand, exhibited asymmetric peaks to a certain extent on all stationary phases, while the elution time was not affected. The developed SEC method was shown to have satisfactory performance in terms of linearity, repeatability, range, and accuracy and exhibit very narrow distributions of elution time and peak symmetry when testing the 138 antibodies indicating its generic performance.

Evaluating the advantages of higher heat conductivity in a recently developed type of core-shell diamond stationary phase particle in UHPLC.

In recent studies, the nature and magnitude of the temperature gradients developed in ultra-high pressure liquid chromatography (UHPLC), were found to be dependent on the heat conductivity properties of the column matrices, but also, on the principle used for controlling the temperature over the column. Here, we investigated the potential of using highly heat conductive diamond-based stationary phases (85 times higher than silica), for reducing the temperature gradients. The stationary phases investigated were a (i) Diamond Analytics FLARE column, based on particles comprised of a graphite core surrounded by a very thin diamond shell, and two silica hybrid columns: (ii) a core-shell silica Kromasil Eternity Shell column and (iii) a fully porous silica Kromasil Eternity XT column. Models were developed based on two-dimensional heat transfer theory and mass transfer theory, which were used to model the temperature profiles and the migration of an analyte band accounting for column efficiencies at different flow rates. For the silica-based columns, using water-controlled temperature mode, the temperature gradients along the column axes are suppressed whereas temperature gradients in the radial direction prevails resulting in decreased column efficiencies. Using these columns with air-controlled temperature mode, the radial temperature gradients are reduced whereas temperature gradients along the column prevails resulting in decreased retention times. With the Diamond FLARE column, there was no loss in column efficiency using the water-controlled temperature mode and the van Deemter curves are almost identical using both temperature control modes. Thus, for the Diamond FLARE column, in contrast to the silica-based columns, there are almost no losses of column efficiencies due to reduced radial temperature gradients independent on how the column temperature was controlled.

Determining gradient conditions for peptide purification in RPLC with machine-learning-based retention time predictions.

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning-based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10-50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on average, 2.6 times larger. This indicates that the model needs to be recalibrated when changing the TFA concentration for the CSH column. Studying possible scale-up complications from UHPLC to HPLC such as pressure, viscous heating (i.e., temperature gradients), and stationary-phase properties (e.g., packing heterogeneity and surface chemistry) revealed that all these factors were minor to negligible. The pressure effect had the largest effect on the retention, but increased retention by only 3%. In the presented case, method development can therefore proceed using UHPLC and then be robustly transferred to HPLC.

A practical approach for predicting retention time shifts due to pressure and temperature gradients in ultra-high-pressure liquid chromatography.

Large pressure gradients are generated in ultra-high-pressure liquid chromatography (UHPLC) using sub-2µm particles causing significant temperature gradients over the column due to viscous heating. These pressure and temperature gradients affect retention and ultimately result in important selectivity shifts. In this study, we developed an approach for predicting the retention time shifts due to these gradients. The approach is presented as a step-by-step procedure and it is based on empirical linear relationships describing how retention varies as a function of temperature and pressure and how the average column temperature increases with the flow rate. It requires only four experiments on standard equipment, is based on straightforward calculations, and is therefore easy to use in method development. The approach was rigorously validated against experimental data obtained with a quality control method for the active pharmaceutical ingredient omeprazole. The accuracy of retention time predictions was very good with relative errors always less than 1% and in many cases around 0.5% (n=32). Selectivity shifts observed between omeprazole and the related impurities when changing the flow rate could also be accurately predicted resulting in good estimates of the resolution between critical peak pairs. The approximations which the presented approach are based on were all justified. The retention factor as a function of pressure and temperature was studied in an experimental design while the temperature distribution in the column was obtained by solving the fundamental heat and mass balance equations for the different experimental conditions. We strongly believe that this approach is sufficiently accurate and experimentally feasible for this separation to be a valuable tool when developing a UHPLC method. After further validation with other separation systems, it could become a useful approach in UHPLC method development, especially in the pharmaceutical industry where demands are high for robustness and regulatory oversight.

The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography.

The adsorption mechanism for three peptides was studied under overloaded conditions through adsorption isotherm measurements in the presence of an ion-pairing reagent, trifluoroacetic acid (TFA), on an end-capped C18-bonded stationary phase. The overall aim of the study was to obtain a better understanding of how the acetonitrile and the TFA fractions in the eluent affected the overloaded elution profiles and the selectivity between peptides using mechanistic modelling and multivariate design of experiments. When studying the effect of TFA, direct evidence for ion pair formation between a peptide and TFA in acetonitrile-water solutions was provided by fluorine-proton nuclear Overhauser NMR enhancement experiments and the adsorption of TFA on the stationary phase was measured by frontal analysis. The adsorption isotherms for each peptide were then determined by the inverse method at eight TFA concentrations ranging from 2.6mM to 37.3mM (0.02-0.29vol-%) in isocratic elution. The equilibrium between the peptide ion and the peptide-TFA complex was modelled by coupling the mass-balance to reaction kinetics and determining separate adsorption isotherms for the two species. We found that a Langmuir isotherm described the elution profile of peptide-TFA complex well while the peptide ion was described by a bi-Langmuir adsorption isotherm since it exhibited strong secondary interactions. The elution profiles had an unfavorable shape at low TFA concentrations consisting of a spike in their front and a long tailing rear due to the secondary interactions for the peptide ion having very low saturation capacity. The acetonitrile dependence on the adsorption isotherms was studied by determination of adsorption isotherms directly from elution profiles obtained in gradient elution which enabled a broad acetonitrile interval to be studied. Here, it was found that the column saturation capacity was quickly reached at very low acetonitrile fractions and that there were significant variations in adsorption with the molecular weight. Finally, practical implications for method development are discussed based on an experimental design where gradient slope and TFA concentrations are used as factors.

A fundamental study of the impact of pressure on the adsorption mechanism in reversed-phase liquid chromatography.

A fundamental investigation of the pressure effect on individual adsorption sites was undertaken based on adsorption energy distribution and adsorption isotherm measurements. For this purpose, we measured adsorption equilibrium data at pressures ranging from 100 to 1000bar at constant flow and over a wide concentration range for three low-molecular-weight solutes, antipyrine, sodium 2-naphthalenesulfonate, and benzyltriethylammonium chloride, on an Eternity C18 stationary phase. The adsorption energy distribution was bimodal for all solutes, remaining clearly so at all pressures. The bi-Langmuir model best described the adsorption in these systems and two types of adsorption sites were identified, one with a low and another with a high energy of interaction. Evidence exists that the low-energy interactions occur at the interface between the mobile and stationary phases and that the high-energy interactions occur nearer the silica surface, deeper in the C18 layer. The contribution of each type of adsorption site to the retention factor was calculated and the change in solute molar volume from the mobile to stationary phase during the adsorption process was estimated for each type of site. The change in solute molar volume was 2-4 times larger at the high-energy site, likely because of the greater loss of solute solvation layer when penetrating deeper into the C18 layer. The association equilibrium constant increased with increasing pressure while the saturation capacity of the low-energy site remained almost unchanged. The observed increase in saturation capacity for the high-energy site did not affect the column loading capacity, which was almost identical at 50- and 950-bar pressure drops over the column.

A quality control method enhancement concept-Continual improvement of regulatory approved QC methods.

Quality Control methods (QC-methods) play an important role in the overall control strategy for drug manufacturing. However, efficient life-cycle management and continual improvement are hindered due to a variety of post-approval variation legislations across territories and a lack of harmonization of the requirements. As a result, many QC-methods fall behind the technical development. Developing the QC-method in accordance with the Quality by Design guidelines gives the possibility to do continual improvements inside the original Method Operable Design Region (MODR). However, often it is necessary to do changes outside the MODR, e.g. to incorporate new technology that was not available at the time the original method was development. Here, we present a method enhancement concept which allows minor adjustments, within the same measuring principle, outside the original MODR without interaction with regulatory agencies. The feasibility of the concept is illustrated by a case study of a QC-method based on HPLC, assumed to be developed before the introduction of UHPLC, where the switch from HPLC to UHPLC is necessary as a continual improvement strategy. The concept relies on the assumption that the System Suitability Test (SST) and failure modes are relevant for other conditions outside the MODR as well when the same measuring principle is used. It follows that it should be possible to move outside the MODR as long as the SST has passed. All minor modifications of the original, approved QC-method must be re-validated according to a template given in the original submission and a statistical equivalence should be shown between the original and modified QC-methods. To summarize, revalidation is handled within the pharmaceutical quality control system according to internal change control procedures, but without interaction with regulating agencies.

A closer study of peak distortions in supercritical fluid chromatography as generated by the injection.

In SFC the sample cannot be dissolved in the mobile phase, so it is often dissolved in pure modifier, or another liquid, sometimes resulting in serious distortions of the eluted peak profiles already at moderately high injection volumes. It is suspected the reasons for these effects are solvent strength mismatch and/or viscosity mismatch. This study presents a systematic and fundamental investigation of the origin of these peak deformations due to the injection solvent effects in SFC, using both systematic experiments and numerical modeling. The first set of experiments proved that the injection volume and the elution strength of the sample solution had a major impact of the shapes of the eluted peaks. Secondly, the sample band elution profile was numerically modeled on a theoretical basis assuming both un-retained and retained co-solvent injection plugs, respectively. These calculations quantitatively confirmed our first set of experiments but also pointed out that there is also an additional significant effect. Third, viscous fingering experiments were performed using viscosity contrast conditions imitating those encountered in SFC. These experiments clearly proved that viscous fingering effects play a significant role. A new method for determination of adsorption isotherms of solvents was also developed, called the "Retention Time Peak Method" (RTPM). The RTPM was used for fast estimation of the adsorption isotherms of the modifier and required using only two experiments.

Evaluation of scale-up from analytical to preparative supercritical fluid chromatography.

An approach for reliable transfer from analytical to preparative scale supercritical fluid chromatography was evaluated. Here, we accounted for the conditions inside the columns as well as to the fact that most analytical instruments are volume-controlled while most preparative scale units are mass-controlled. The latter is a particular problem when performing pilot scale experiments and optimizations prior to scaling up to production scale. This was solved by measuring the mass flow, the pressure and the temperature on the analytical unit using external sensors. Thereafter, it was revealed with a design of experiments approach that the methanol fraction and the pressure are the two most important parameters to control for preserved retention throughout the scale-up; for preserved selectivity the temperature was most important in this particular system. Using this approach, the resulting chromatograms from the preparative unit agreed well with those from the analytical unit while keeping the same column length and particles size. A brief investigation on how the solute elution volume varies with the volumetric flow rate revealed a complex dependency on pressure, density and apparent methanol content. Since the methanol content is a parameter of great importance to control during the scale up, we must be careful when changing operational and column design conditions which generates deviations in pressure, density and methanol content between different columns.

Method transfer from high-pressure liquid chromatography to ultra-high-pressure liquid chromatography. II. Temperature and pressure effects.

The importance of the generated temperature and pressure gradients in ultra-high-pressure liquid chromatography (UHPLC) are investigated and compared to high-pressure liquid chromatography (HPLC). The drug Omeprazole, together with three other model compounds (with different chemical characteristics, namely uncharged, positively and negatively charged) were used. Calculations of the complete temperature profile in the column at UHPLC conditions showed, in our experiments, a temperature difference between the inlet and outlet of 16 °C and a difference of 2 °C between the column center and the wall. Through van't Hoff plots, this information was used to single out the decrease in retention factor (k) solely due to the temperature gradient. The uncharged solute was least affected by temperature with a decrease in k of about 5% while for charged solutes the effect was more pronounced, with k decreases up to 14%. A pressure increase of 500 bar gave roughly 5% increase in k for the uncharged solute, while omeprazole and the other two charged solutes gave about 25, 20 and 15% increases in k, respectively. The stochastic model of chromatography was applied to estimate the dependence of the average number of adsorption/desorption events (n) and the average time spent by a molecule in the stationary phase (τs) on temperature and pressure on peak shape for the tailing, basic solute. Increasing the temperature yielded an increase in n and decrease in τs which resulted in less skew at high temperatures. With increasing pressure, the stochastic modeling gave interesting results for the basic solute showing that the skew of the peak increased with pressure. The conclusion is that pressure effects are more pronounced for both retention and peak shape than the temperature effects for the polar or charged compounds in our study.

Evaluation of co-solvent fraction, pressure and temperature effects in analytical and preparative supercritical fluid chromatography.

A chemometric approach is used for studying the combined effect of temperature, pressure and co-solvent fraction in analytical and preparative supercritical fluid chromatography (SFC). More specifically, by utilizing design of experiments coupled with careful measurements of the experimental conditions the interaction between pressure, temperature and co-solvent fraction was studied with respect to productivity, selectivity and retention in chiral SFC. A tris-(3,5-dimethylphenyl) carbamoyl cellulose stationary phase with carbon dioxide/methanol as mobile phase and the two racemic analytes trans-stilbene oxide (TSO) and 1,1'-bi-2-naphthol (BINOL) were investigated. It was found for the investigated model system that the co-solvent fraction and pressure were the parameters that most affected the retention factors and that the co-solvent fraction and column temperature were most important for controlling the selectivity. The productivity in the preparative mode of SFC was most influenced by the co-solvent fraction and temperature. Both high co-solvent fraction and temperature gave maximum productivity in the studied design space.

Method transfer from high-pressure liquid chromatography to ultra-high-pressure liquid chromatography. I. A thermodynamic perspective.

This is the first investigation in a series that aims to enhance the scientific knowledge needed for reliable analytical method transfer between HPLC and UHPLC using the quality by design (QbD) framework. Here, we investigated the differences and similarities from a thermodynamic point of view between RP-LC separations conducted with 3.5µm (HPLC) and 1.7µm (UHPLC) C18 particles. Three different model solutes and one pharmaceutical compound were used: the uncharged cycloheptanone, the cationic benzyltriethylammonium chloride, the anionic sodium 2-naphatlene sulfonate and the pharmaceutical compound omeprazole, which was anionic at the studied pH. Adsorption data were determined for the four solutes at varying fractions of organic modifier and in gradient elution in both the HPLC and UHPLC system, respectively. From the adsorption data, the adsorption energy distribution of each compound was calculated and the adsorption isotherm model was estimated. We found that the adsorption energy distribution was similar, with only minor differences in degree of homogeneity, for HPLC and UHPLC stationary phases. The adsorption isotherm model did not change between HPLC and UHPLC, but the parameter values changed considerably especially for the ionic compounds. The dependence of the organic modifier followed the same trend in HPLC as in UHPLC. These results indicates that the adsorption mechanism of a solute is the same on HPLC and UHPLC stationary phases which simplifies design of a single analytical method applicable to both HPLC and UHPLC conditions within the QbD framework.

Fast estimation of adsorption isotherm parameters in gradient elution preparative liquid chromatography II: the competitive case.

Experimental competitive adsorption isotherms were successfully determined directly from overloaded elution profiles in gradient elution mode using an extended inverse method. This approach differs from the existing methods in one important aspect - no isocratic experiments are necessary which makes it possible to study adsorption of substances whose retention factors vary strongly with the mobile-phase composition. The approach was verified with simulated binary data and with experimental data from gradient separations of a cyclohexanone/cycloheptanone mixture. For the synthetic data, the original adsorption isotherm parameters were found using a two-step estimation procedure. In the first step analytical peaks were used to estimate the "analytical" part of the Langmuir equation and in the second step the association equilibrium parameters were estimated from two simulated overloaded elution profiles. For the experimental data, a three-step approach was used. The two first steps were used to reduce the calculation time so that parameter estimation could be performed on an ordinary computer. In the first step, analytical peaks were used to estimate the "analytical" part of the bi-Langmuir equation. In the second step, initial guesses for all other parameters were determined separately for each solute using the faster Rouchon algorithm. In the final and third step, the more accurate orthogonal collocation on finite elements algorithm, was used to fine-tune the isotherm parameters. The model could accurately predict the shape of overloaded elution profiles. The shape of the adsorption isotherms agreed well with those determined with the standard isocratic method, although the numerical values were not the same. The extended inverse method is well suited for process optimization where few experiments and accurate predictions are important.

Fast estimation of adsorption isotherm parameters in gradient elution preparative liquid chromatography. I: the single component case.

The inverse method is a numeric method for fast estimation of adsorption isotherm parameters directly from overloaded elution profiles. However, it has previously only been used for isocratic experiments. Here we will extend the inverse method so it can be used for gradient elution too. This extended inverse method will make it possible to study the adsorption of substances whose retention factor vary strongly with the mobile-phase composition, like peptides and proteins, where the classic methods will fail. Our extended inverse method was verified using both simulations and real experiments. For simulated overloaded elution profiles we were able to determine almost exact Langmuir adsorption isotherm parameters with the new approach. From real experimental data, bi-Langmuir adsorption parameters were estimated using both the perturbation peak method and the extended inverse method. The shape of the acquired adsorption isotherms did match over the considered concentration range; however, the adsorption isotherm parameters found with the two methods were not the same. This is probably due to the fact that adsorption isotherm estimated with the inverse method is only a good approximation up to the highest eluted concentration in the used chromatograms. But this is not a serious drawback from a process point of view where the main objective is to make accurate predictions of elution profiles. The bi-Langmuir adsorption isotherm obtained with both methods could accurately predict the shape of overloaded elution profiles.