| 1. |
- Bagge, Joakim, et al.
(author)
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Impact of stationary-phase pore size on chromatographic performance using oligonucleotide separation as a model
- 2020
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In: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1634, s. 1-10
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Journal article (peer-reviewed)abstract
- A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 angstrom rather than 100 angstrom pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.
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| 2. |
- Encarnacao, Joao Crispim, et al.
(author)
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Impact of assay temperature on antibody binding characteristics in living cells : A case study
- 2017
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In: BIOMEDICAL REPORTS. - : Spandidos Publications. - 2049-9434 .- 2049-9442. ; 7:5, s. 400-406
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Journal article (peer-reviewed)abstract
- Kinetic and thermodynamic studies of ligand-receptor interactions are essential for increasing the understanding of receptor activation mechanisms and drug behavior. The characterization of molecular interactions on living cells in real-time goes beyond most current binding assays, and provides valuable information about the dynamics and underlying mechanism of the molecules in a living system. The effect of temperature on interactions in cell-based assays is, however, rarely discussed. In the present study, the effect of temperature on binding of monoclonal antibodies, cetuximab and pertuzumab to specific receptors on living cancer cells was evaluated, and the affinity and kinetics of the interactions were estimated at selected key temperatures. Changes in the behavior of the interactions, particularly in the on- and off-rates were observed, leading to greatly extended time to reach the equilibrium at 21 degrees C compared with at 37 degrees C. However, the observed changes in kinetic characteristics were less than a factor of 10. It was concluded that it is possible to conduct real-time measurements with living cells at different temperatures, and demonstrated that influences of the ambient temperature on the interaction behavior are likely to be less than one order of magnitude.
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| 3. |
- Enmark, Martin, 1984-, et al.
(author)
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A Retention-Matching Strategy for Method Transfer in Supercritical Fluid Chromatography : Introducing the Isomolar Plot Approach
- 2021
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:16, s. 6385-6393
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Journal article (peer-reviewed)abstract
- A strategy to match any retention shifts due to increased or decreased pressure drop during supercritical fluid chromatography (SFC) method transfer is presented. The strategy relies on adjusting the co-solvent molarity without the need to adjust the back-pressure regulator. Exact matching can be obtained with minimal changes in separation selectivity. To accomplish this, we introduce the isomolar plot approach, which shows the variation in molar co-solvent concentration depending on the mass fraction of co-solvent, pressure, and temperature, here exemplified by CO2-methanol. This plot allowed us to unify the effects of the co-solvent mass fraction and density on retention in SFC. The approach, which was verified on 12 known empirical retention models for each enantiomer of six basic pharmaceuticals, allowed us to numerically calculate the apparent retention factor for any column pressure drop. The strategy can be implemented either using a mechanistic approach if retention models are known or empirically by iteratively adjusting the co-solvent mass fraction. As a rule of thumb for the empirical approach, we found that the relative mass fraction adjustment needed is proportional to the relative change in the retention factor caused by a change in the pressure drop. Different proportionality constants were required to match retention in the case of increasing or decreasing pressure drops.
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| 4. |
- Enmark, Martin, et al.
(author)
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A systematic investigation of algorithm impact in preparative chromatography with experimental verifications
- 2011
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In: Journal of Chromatography A. - Amsterdam : Elsevier. - 0021-9673 .- 1873-3778. ; 1218:5, s. 662-672
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Journal article (peer-reviewed)abstract
- Computer-assisted optimization of chromatographic separations requires finding the numerical solution of the Equilibrium-Dispersive (ED) mass balance equation. Furthermore, the competitive adsorption isotherms needed for optimization are often estimated numerically using the inverse method that also solves the ED equations. This means that the accuracy of the estimated adsorption isotherm parameters explicitly depends on the numerical accuracy of the algorithm that is used to solve the ED equations. The fast and commonly used algorithm for this purpose, the Rouchon Finite Difference (RFD) algorithm, has often been reported not to be able to accurately solve the ED equations for all practical preparative experimental conditions, but its limitations has never been completely and systematically investigated. In this study, we thoroughly investigate three different algorithms used to solve the ED equations: the RFD algorithm, the Orthogonal Collocation on Finite Elements (OCFE) method and a Central Difference Method (CDM) algorithm, both for increased theoretical understanding and for real cases of industrial interest. We identified discrepancies between the conventional RFD algorithm and the more accurate OCFE and CDM algorithms for several conditions, such as low efficiency, increasing number of simulated components and components present at different concentrations. Given high enough efficiency, we experimentally demonstrate good prediction of experimental data of a quaternary separation problem using either algorithm, but better prediction using OCFE/CDM for a binary low efficiency separation problem or separations when the compounds have different efficiency. Our conclusion is to use the RFD algorithm with caution when such conditions are present and that the rule of thumb that the number of theoretical plates should be greater than 1000 for application of the RFD algorithm is underestimated in many cases
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| 5. |
- Enmark, Martin, et al.
(author)
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A systematic investigation on the accuracy of computer simulations for optical isomers in industrial settings
- 2010
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Conference paper (peer-reviewed)abstract
- Predicting the band profiles of typical chromatographic separations using the approach of the inverse method (IM) [2] requires finding the numerical solution of the Equilibrium-Dispersive (ED) mass balance equation [1]. The accuracy of the determination of the competitive adsorption isotherms is therefore explicitly dependent on the choice of algorithm to solve the ED model. Earlier studies have shown that the well known RFD algorithm [3] has limitations in its accuracy for certain simulated conditions such as column efficiency and adsorption isotherm type [5]. In this study, we thoroughly investigate three different algorithms, the rapid and well known Rouchon Finite Difference (RFD) algorithm [3], the Orthogonal Collocation on Finite Elements (OCFE) method [4] and the Central Difference Method (CDM) algorithm developed by us. Firstly, a systematic investigation is made, comparing the different algorithms under a broad range of different synthetic conditions. Secondly, we apply all three algorithms using the IM on real experimental systems; (I) one high efficiency quaternary separation and (II) one pharmaceutical industry application with a chiral intermediate separation. We have found discrepancies between the conventional (RFD) and the more accurate (OCFE and CDM) algorithms for several synthetic conditions such as low efficiency, increasing number of simulated components and components present at different concentrations. Given high enough efficiency, we experimentally demonstrate good prediction of experimental data of a quaternary separation problem using either algorithm but better prediction of OCFE/CDM for a binary low efficiency separation problem. Our conclusion is to use the RFD algorithm with caution when any of the particular conditions we have investigated is valid. When the highest accuracy is sought, there is no doubt that OCFE or CDM should be the algorithms of choice. However, given the computational speed of RFD, we also recommend it for preliminary parameter fitting. For conditions where we have shown the algorithms to produce practically identical solutions, RFD can be applied with greater certainty. It remains to be thoroughly investigated the effect on the particular shape of the adsorption isotherm on the obtained solution, for example type III having inflection points.
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| 6. |
- Enmark, Martin, 1984-, et al.
(author)
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Analytical and preparative separation of phosphorothioated oligonucleotides : Columns and ion-pair reagents
- 2020
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In: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 412:2, s. 299-309
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Journal article (peer-reviewed)abstract
- Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 520mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.
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| 7. |
- Enmark, Martin, 1984-, et al.
(author)
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Building machine-learning-based models for retention time and resolution predictions in ion pair chromatography of oligonucleotides
- 2022
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In: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1671
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Journal article (peer-reviewed)abstract
- Support vector regression models are created and used to predict the retention times of oligonucleotides separated using gradient ion-pair chromatography with high accuracy. The experimental dataset consisted of fully phosphorothioated oligonucleotides. Two models were trained and validated using two pseudo orthogonal gradient modes and three gradient slopes. The results show that the spread in retention time differs between the two gradient modes, which indicated varying degree of sequence dependent separation. Peak widths from the experimental dataset were calculated and correlated with the guanine cytosine content and retention time of the sequence for each gradient slope. This data was used to predict the resolution of the n - 1 impurity among 250 0 0 0 random 12-and 16-mer sequences; showing one of the investigated gradient modes has a much higher probability of exceeding a resolution of 1.5, particularly for the 16-mer sequences. Sequences having a high guanine-cytosine content and a terminal C are more likely to not reach critical resolution. The trained SVR models can both be used to identify characteristics of different separation methods and to assist in the choice of method conditions, i.e. to optimize resolution for arbitrary sequences. The methodology presented in this study can be expected to be applicable to predict retention times of other oligonucleotide synthesis and degradation impurities if provided enough training data.
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| 8. |
- Enmark, Martin, et al.
(author)
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Computer-assisted optimization in preparative SFC, Challenges, Pitfalls and Solutions
- 2010
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Conference paper (peer-reviewed)abstract
- Preparative packed column SFC (pSFC) is an environmentally favorable alternative to normal phase HPLC. We are participating in a larger research project where the overreaching goal is to make an impact in the recovery of high-value compounds from agricultural byproducts and waste materials using environmentally sustainable techniques. Our specific task is to isolate large amounts of valuable compounds from discarded biomaterials using pSFC. It is still not possible to accurately model, simulate and optimize a preparative SFC proess, although SFC has been in use for almost fifty years. This is mainly because of the compressibility of the mobile phase and how this influences the physical parameters of the system. More particular, if pSFC is operated under combinations of back pressures and flow rates that give negligible pressure drop, it has been demonstrated and implied, that the tools developed and used within HPLC, can be transferred and used successfully [1, 2]. However, when the pressure drop becomes noticeable, the induced density gradient significantly increases the complexity. An earlier study has clearly described the effect of this pressure gradient on the retention factor, and also the column efficiency [3]. However, it remains to be investigated how the entire adsorption isotherm depends on the density.In this study we will investigate the use of the inverse and the perturbation peak methods, originally developed within LC, to determine the nonlinear adsorption isotherm for i) negligible, and ii) non-negligible pressure drop conditions respectively. To do this, we will use a simple racemic substance and a cellulose based CSP with a column equipped with pressure sensors at the inlet and outlet. Using obtained parameters we will validate the methods by simulating the separation using a variable coefficient version of the Equilibrium-Dispersive model for cases i and ii. [1] S. Ottiger, J. Kluge, A. Rajendran , M. Mazzotti, J. Chromatogr., A 1162 (2007) 74-82[2] C. Wenda, A. Rajendran, J. Chromatogr., A 1216 (2009) 8750-8758[3]A. Rajendran, O. Kräuchi, M. Mazzotti, M. Morbidelli, J Chromatogr., A, 1092 (2005) 149-160[4] G. Guiochon, A. Felinger, D. G. Shirazi, A. M. Katti, Fundamentals of Preparative and Nonlinear Chromatography 2nd Ed, Elsevier Academic Press (2006).
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| 9. |
- Enmark, Martin, et al.
(author)
-
Computer Assisted Optimization of Pharmaceutical Purification - The Impact of Algorithms and Experimental Approach
- 2010
-
Conference paper (peer-reviewed)abstract
- Computer assisted process optimization of chromatographic separations requires the selection of computer algorithms and measurement of relevant parameters. In this study, we will investigate how the choice of algorithms and the number of performed initial experiments affect the estimated optimal separations conditions. We will focus on conditions typically encountered for slurry packing coated 20 µm CSP in large-scale columns used for pharmaceutical intermediate purification.Probably the most essential for parameters in this context are the adsorption isotherm parameters for the components. The rapid "inverse method" is commonly used and this method requires that one numerically solve the mass balance equations describing the chromatographic process. Here we thoroughly investigate how different algorithms that solve the Equilibrium-Dispersive (ED) mass balance equations will affect the estimated adsorption isotherm parameters.Furthermore, we will investigate and compare how different strategies affect the prediction of the optimal separation conditions. First, we will use a more rapid approach that requires a minimum of experiments and uses standard algorithms to estimate optimal conditions. Secondly, we will use a more exact approach that requires more experiments and uses more advanced simulation- and optimization algorithms
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| 10. |
- Enmark, Martin, 1984-, et al.
(author)
-
Development of a unified gradient theory for ion-pair chromatography using oligonucleotide separations as a model case
- 2023
-
In: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1691
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Journal article (peer-reviewed)abstract
- Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield and productivity while consuming less co-solvent.
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