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Characterizing Chromatography Media: NMR-based Approaches
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.ORCID iD: 0000-0001-6614-5710
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Liquid chromatography is an essential technique in manufacturing biopharmaceuticals where it is used on all scales from analytical applications in R&D to full-scale production. In chromatography the target molecule, typically a protein, is separated and purified from other components and contaminants. Separation is based on different affinities of different molecules for the chromatographic medium and the physical and chemical properties of the latter determine the outcome. Controlling and designing those properties demand efficient analytical techniques.

In this thesis the approach was to develop characterization methods based on nuclear magnetic resonance (NMR) spectroscopy for the assessment of various important physico-chemical properties. The rationale behind this strategy was that the versatility of NMR – with its chemical and isotopic specificity, high dynamic range, and direct proportionality between the integral intensity of the NMR signal and the concentration of spin-bearing atomic nuclei (e.g., 1H, 13C, 31P and 15N) – often renders it a very good choice for both qualitative and quantitative evaluations.

These characteristics of NMR enabled us to develop two quantification methods for chromatography-media ligands, the functional groups that provide the specific interactions for the molecules being separated. Furthermore, a new method for measuring the distribution of macromolecules between the porous chromatographic beads and the surrounding liquid was established. The method, which we have named size-exclusion quantification (SEQ) NMR, utilizes the fact that it is possible to assess molecular size distribution from corresponding distribution of the molecular self-diffusion coefficient where the latter is accessible by NMR. SEQ-NMR results can also be interpreted in terms of pore-size distribution within suitable models. Finally, we studied self-diffusion of small molecules inside the pores of chromatographic beads. The results provided new insights into what affects the mass transport in such systems.

The methods presented in this thesis are accurate, precise, and in many aspects better than conventional ones in terms of speed, sample consumption, and potential for automation. They are thus important tools that can assist a better understanding of the structure and function of chromatography media. In the long run, the results in this project may lead, via better chromatographic products, to better drugs and improved health.

Abstract [sv]

Vätskekromatografi är en viktig teknik för tillverkning av biologiska läkemedel och används för alltifrån småskaliga analytiska applikationer till fullskalig produktion. I kromatografi separeras och renas målmolekylen (oftast ett protein), från andra komponenter och föroreningar genom att utnyttja molekylernas olika affinitet för det kromatografiska mediumet, vars fysikaliska och kemiska egenskaper har stor betydelse för hur separationen fungerar. För att kunna kontrollera och designa dessa egenskaper krävs effektiva analysmetoder.

Strategin i den här avhandlingen var att utveckla metoder baserade på kärnmagnetisk resonans (NMR) spektroskopi för att karaktärisera flera viktiga fysikalisk-kemiska egenskaper. Anledningen till denna strategi är att mångsidigheten hos NMR – med dess kemiska och isotopiska specificitet, stora dynamiska omfång och direkta proportionalitet mellan NMR-signalens integralintensitet och koncentrationen av spinnbärande atomkärnor (t.ex. 1H, 13C, 31P och 15N) - ofta gör den till det bästa valet för både kvalitativa och kvantitativa tillämpningar.

Dessa egenskaper hos NMR gjorde att vi kunde utveckla två kvantifieringsmetoder för kromatografimedia-ligander, dvs de funktionella grupperna som ger de specifika interaktioner som gör att molekylerna kan separeras. Dessutom har en ny metod för att mäta fördelningen av makromolekyler mellan de porösa kromatografiska pärlorna och den omgivande vätskan tagits fram. Metoden, som vi har valt att kalla size-exclusion quantification (SEQ) NMR, utnyttjar det faktum att det är möjligt att mäta molekylstorleksfördelningen genom att mäta motsvarande fördelning av självdiffusionskoefficienter, där den sistnämnda kan bestämmas med NMR. Resultaten från SEQ-NMR kan tolkas i termer av porstorleksfördelningar genom att använda lämpliga modeller. Slutligen studerade vi självdiffusion av små molekyler inuti porerna i kromatografiska pärlor. Resultaten gav nya insikter om vad som påverkar masstransporten i sådana system.

De metoder som presenteras i denna avhandling är noggranna, precisa och på många sätt bättre än konventionella metoder när det gäller hastighet, låg provförbrukning och automatiseringspotential. De nya metoderna är därför viktiga verktyg som kan hjälpa till att ge en bättre förståelse av struktur och funktion hos kromatografimedia. I det långa loppet kan resultat från det här projektet kunna bidra till effektivare kromatografiska produkter, vilket i slutändan kan leda till bättre läkemedel och hälsa.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , 61 p.
Series
TRITA CHE Report, ISSN 1654-1081 ; 2017:20
Keyword [en]
chromatography, nuclear magnetic resonance, NMR, self-diffusion, magic-angle spinning, quantitative, distribution coefficient, pore size
National Category
Physical Chemistry
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-204572ISBN: 978-91-7729-334-7 OAI: oai:DiVA.org:kth-204572DiVA: diva2:1085281
Public defence
2017-05-04, F3, Lindstedtsvägen 26, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council
Note

QC 20170403

Available from: 2017-04-03 Created: 2017-03-28 Last updated: 2017-04-03Bibliographically approved
List of papers
1. C-13 SPE MAS measurement of ligand concentration in compressible chromatographic beads
Open this publication in new window or tab >>C-13 SPE MAS measurement of ligand concentration in compressible chromatographic beads
2015 (English)In: Magnetic Resonance in Chemistry, ISSN 0749-1581, E-ISSN 1097-458X, Vol. 53, no 8, 572-577 p.Article in journal (Refereed) Published
Abstract [en]

A method for measuring the ligand concentration in heterogeneous materials like chromatography media is described. In this method, C-13 single pulse excitation magic angle spinning NMR experiment with broadband H-1 decoupling is used to determine the peak integrals for a butyl ligand in the spectrum of a dried chromatography medium. Within a carefully controlled protocol, those integrals compared with that of the internal reference compound dimethyl sulfone provide the required volume concentration with an accuracy of ca 2%. The effects of temperature, degree of hydration, and other experimental parameters are discussed. Copyright (C) 2015 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.

Keyword
NMR, C-13, solid-state NMR, quantitative NMR, agarose, pore
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-173449 (URN)10.1002/mrc.4257 (DOI)000359802300003 ()26053054 (PubMedID)2-s2.0-84946492473 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150915

Available from: 2015-09-15 Created: 2015-09-11 Last updated: 2017-03-28Bibliographically approved
2. High-resolution magic angle spinning H-1 NMR measurement of ligand concentration in solvent-saturated chromatographic beads
Open this publication in new window or tab >>High-resolution magic angle spinning H-1 NMR measurement of ligand concentration in solvent-saturated chromatographic beads
2016 (English)In: Magnetic Resonance in Chemistry, ISSN 0749-1581, E-ISSN 1097-458X, Vol. 54, no 4, 291-297 p.Article in journal (Refereed) Published
Abstract [en]

A method based on H-1 high-resolution magic angle spinning NMR has been developed for measuring concentration accurately in heterogeneous materials like that of ligands in chromatography media. Ligand concentration is obtained by relating the peak integrals for a butyl ligand in the spectrum of a water-saturated chromatography medium to the integral of the added internal reference. The method is fast, with capacity of 10min total sample preparation and analysis time per sample; precise, with a reproducibility expressed as 1.7% relative standard deviation; and accurate, as indicated by the excellent agreement of derived concentration with that obtained previously by C-13 single-pulse excitation MAS NMR. The effects of radiofrequency field inhomogeneity, spin rate, temperature increase due to spinning, and distribution and re-distribution of medium and reference solvent both inside the rotor during spinning and between bulk solvent and pore space are discussed in detail.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2016
Keyword
H-1, HR-MAS NMR, quantitative NMR, heterogeneous material, agarose, tissue
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-185057 (URN)10.1002/mrc.4370 (DOI)000372298200002 ()26791865 (PubMedID)2-s2.0-84959509396 (Scopus ID)
Note

QC 20160415

Available from: 2016-04-15 Created: 2016-04-11 Last updated: 2017-03-28Bibliographically approved
3. SEQ-NMR: A new tool for measuring distribution coefficients and pore size in chromatography media
Open this publication in new window or tab >>SEQ-NMR: A new tool for measuring distribution coefficients and pore size in chromatography media
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a new method, SEQ-NMR (size-exclusion quantification NMR spectroscopy) for measuring equilibrium distribution coefficients, Keq, in chromatography media. The same method is also generally applicable to measure pore sizes in any porous media. Keq versus probe size, the selectivity curve, is obtained by equilibrating a chromatographic medium with a solution of polymers with a broad size distribution. The solution before and after equilibration is analyzed with NMR diffusion experiments. The joint least-squares analysis of the two diffusion attenuation curves under suitable constraints provides the extent by which the different polymer fractions are diluted upon equilibration that in turn yields the selectivity curve. The relation of the applied procedure to Inverse Laplace transformation is clarified. Simulations probe the performance of the method, in particular with respect to signal-to-noise ratio (SNR) and other parameters used in the data inversion process. The method is demonstrated experimentally using dextran polymers and the chromatographic medium Sephacryl™ S-200 High Resolution for which it yields, within a cylindrical pore model, 6.9 nm for the average hydrated pore size. Comparison is made to the analysis of the same system with inverse size-exclusion chromatography (ISEC). Advantages of the method, primarily that in speed, potential for automation, and small sample size, are discussed.

Keyword
nuclear magnetic resonance, NMR, chromatography, distribution coefficient, partition coefficient, diffusion, pore size
National Category
Physical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-204568 (URN)
Funder
Swedish Research Council
Note

QC 20170418

Available from: 2017-03-28 Created: 2017-03-28 Last updated: 2017-04-18Bibliographically approved
4. Diffusive transport in pores. Tortuosity and molecular interaction with the pore wall
Open this publication in new window or tab >>Diffusive transport in pores. Tortuosity and molecular interaction with the pore wall
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The self-diffusion of neat water, dimethylsulfoxide (DMSO), octanol and the molecular components in a water-DMSO solution were measured by 1H and 2H NMR diffusion experiments for those fluids imbibed into Controlled Pore Glasses (CPG). Their highly interconnected structure is scaled by pore size and shows the some pore topology independently of the size. The nominal pore diameter of the explored CPGs varied from 7.5 nm to 72.9 nm. Hence, the ∼μm mean-square diffusional displacement during the explored diffusion times was much larger than the individual pore size. Great care was taken to establish the actual pore volumes of the CPGs. In addition, transverse relaxation experiments processed by Inverse Laplace Transformation were performed to verify that the liquids explored filled exactly the available pore volume. Relative to the respective diffusion coefficients obtained in bulk phases, we observe a reduction in the diffusion coefficient that is independent of pore sizes for the larger pores and becomes larger towards the smaller pores. Geometric tortuosity governs the behavior at larger pore sizes while the interaction with pore walls becomes the dominant factor at our smallest pore diameter. The interaction with the pore wall is not just simple steric one but must in part be dependent on the specific features of the molecules explored here.

Keyword
nuclear magnetic resonance, NMR, self-diffusion, interaction, pore, controlled pore glass
National Category
Physical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-204569 (URN)
Funder
Swedish Research Council
Note

QC 20170418

Available from: 2017-03-28 Created: 2017-03-28 Last updated: 2017-04-18Bibliographically approved

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