| 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. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- Enmark, Martin, 1984-, et al.
(author)
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Development of a unified gradient theory for ion-pair chromatography using oligonucleotide separations as a model case
- 2023
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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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| 6. |
- Enmark, Martin, 1984-, et al.
(author)
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Selectivity limits of and opportunities for ion pair chromatographic separation of oligonucleotides
- 2021
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In: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1651
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Journal article (peer-reviewed)abstract
- A B S T R A C T Here it was investigated how oligonucleotide retention and selectivity factors are affected by electrostatic and non-electrostatic interactions in ion pair chromatography. A framework was derived describing how selectivity depends on the electrostatic potential generated by the ion-pair reagent concentration, co-solvent volume fraction, charge difference between the analytes, and temperature. Isocratic experiments verified that, in separation problems concerning oligonucleotides of different charges, selectivity increases with increasing surface potential and analyte charge difference and with decreasing co-solvent volume fraction and temperature. For analytes of the same charge, for example, diastereomers of phosphorothioated oligonucleotides, selectivity can be increased by decreasing the co-solvent volume fraction or the temperature and has only a minor dependency on the ion-pairing reagent concentration. An important observation is that oligonucleotide retention is driven predominantly by electrostatic interaction generated by the adsorption of the ion-pairing reagent. We therefore compared classical gradient elution in which the co-solvent volume fraction increases over time versus gradient elution with a constant co-solvent volume fraction but with decreasing ion-pair reagent concentration over time. Both modes decrease the electrostatic potential. Oligonucleotide selectivity was found to increase with decreasing ion pairing reagent concentration. The two elution modes were finally applied to two different model anti sense oligonucleotide separation problems, and it was shown that the ion-pair reagent gradient increases the selectivity of non-charge & ndash;based separation problems while maintaining charge-difference & ndash;based selectivity. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
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| 7. |
- Fornstedt, Torgny, 1957-, et al.
(author)
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Chapter 23 - Modeling of preparative liquid chromatography
- 2023. - 3
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In: Liquid Chromatography. - : Elsevier. - 9780323999687 ; , s. 603-624
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Book chapter (other academic/artistic)abstract
- Preparative chromatography is the best generic method currently available for purifying small drugs and valuable chemical components at the 10-kg level. Progress in computer technology, the development of new non-chiral/chiral stationary phases, and numerous improvements in reliability and economic performance have considerably increased the interest in modeling in academia and industry. This chapter introduces the modeling of preparative liquid chromatography in order to improve the purification process for valuable chemical components such as drugs and chiral components. We review the most important column and adsorption models and the methods for determining the essential thermodynamic adsorption data for both column characterization and process improvement. We also cover important operational modes (e.g., separation in gradient mode), cases involving additives or ion-pair reagents, and operational conditions sometimes neglected in the modeling process, for example, involving the impact of injection profiles.
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| 8. |
- Fornstedt, Torgny, 1957-, et al.
(author)
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Method transfer in SFC from a fundamental perspective
- 2022
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In: TrAC. Trends in analytical chemistry. - : Elsevier. - 0165-9936 .- 1879-3142. ; 149
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Journal article (peer-reviewed)abstract
- The fundamental aspects to be considered during method transfer in supercritical fluid chromatography (SFC) are reviewed. The review is limited to mobile phases, stationary phases, and operating conditions generally encountered in current practice. First, the fundamentals of retention in SFC will be explored in relation to fluid composition, co-solvent adsorption to the stationary phase, pressure, and temperature. Second, considerations regarding predictable method transfer will be discussed in relation to instrumentation, columns, retention shifts, and method robustness. This review is not intended to be comprehensive but rather to highlight important issues for understanding and performing reliable method transfer and to give practical guidelines relating to the fundamentals covered.
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| 9. |
- Fornstedt, Torgny, 1957-, et al.
(author)
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Separation of therapeutic oligonucleotides using ion-pair reversed-phase chromatography based on fundamental separation science
- 2023
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In: Journal of Chromatography Open. - : Elsevier. - 2772-3917. ; 3
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Journal article (peer-reviewed)abstract
- In recent decades, there has been a trend toward using larger biological molecules as new active pharmaceutical ingredients (APIs) instead of the classical small organic API molecules. More recently, this trend has shifted from very large biomolecules toward intermediate-sized APIs, such as oligonucleotide therapeutics. Because of their fundamental role in gene regulation, therapeutic oligonucleotides can be directed against their specific ribonucleic acid (RNA) targets, representing a promising customized approach for the treatment of hitherto incurable diseases. There are several FDA-approved oligonucleotide-based therapeutics and many more are awaiting approval. The complicated synthesis and degradation pathways of oligonucleotides, involving sophisticated new chemical modifications, generate hundreds of impurities, in contrast to classical small APIs, which typically contain only around three to five well-defined impurities (Fig. 1). Therefore, this new class of putative drugs entails challenging separation tasks: for example, a small mass change such as 1 Da must be distinguished in a 10,000 Da parent molecule for purposes of both quantification and purification and at extremely high resolution. All therapeutic oligonucleotides must be chemically modified before entering the body. One such modification is the phosphorothioate (PS) modification, which generates diastereomers: for a 20-nucleotide-long PS oligonucleotide, this exceeds half a million diastereomers. In this review, we will examine recently published ion-pair liquid chromatographic separation strategies to meet current challenges in oligonucleotide separations. Ion-exchange chromatography will be briefly discussed based on its merits for large-scale purification. The review focuses on studies combining theory and practice and aiming at the analysis and preparative separation necessary for performing reliable quality control as well as purification. All relevant aspects of the separation systems will be discussed, including the stationary phase, pore size, mobile phase, and ion-pairing reagents. We will also discuss how the properties of the oligonucleotide and its impurities can be exploited to increase separation selectivity. A particular focus will be on the adsorption of ion-pairing reagent and the electrostatic surface potential it generates, allowing for interaction with the highly charged oligonucleotides. Furthermore, the effects of various gradient modes to decrease the electrostatic potential and thereby elute oligonucleotides will be covered.
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| 10. |
- Forssén, Patrik, 1966-, et al.
(author)
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Advanced Analysis of Biosensor Data for SARS-CoV-2 RBD and ACE2 Interactions
- 2020
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:17, s. 11520-11524
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Journal article (peer-reviewed)abstract
- The traditional approach for analyzing interaction data from biosensors instruments is based on the simplified assumption that also larger biomolecules interactions are homogeneous. It was recently reported that the human receptor angiotensin-converting enzyme 2 (ACE2) plays a key role for capturing SARS-CoV-2 into the human target body, and binding studies were performed using biosensors techniques based on surface plasmon resonance and bio-layer interferometry. The published affinity constants for the interactions, derived using the traditional approach, described a single interaction between ACE2 and the SARS-CoV-2 receptor binding domain (RBD). We reanalyzed these data sets using our advanced four-step approach based on an adaptive interaction distribution algorithm (AIDA) that accounts for the great complexity of larger biomolecules and gives a two-dimensional distribution of association and dissociation rate constants. Our results showed that in both cases the standard assumption about a single interaction was erroneous, and in one of the cases, the value of the affinity constant K-D differed more than 300% between the reported value and our calculation. This information can prove very useful in providing mechanistic information and insights about the mechanism of interactions between ACE2 and SARS-CoV-2 RBD or similar systems.
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