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High-resolution magic angle spinning H-1 NMR measurement of ligand concentration in solvent-saturated chromatographic beads
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. GE Healthcare Biosci AB, Sweden.ORCID iD: 0000-0001-6614-5710
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.ORCID iD: 0000-0002-0231-3970
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
Resource type
Text
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. Vol. 54, no 4, 291-297 p.
Keyword [en]
H-1, HR-MAS NMR, quantitative NMR, heterogeneous material, agarose, tissue
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-185057DOI: 10.1002/mrc.4370ISI: 000372298200002PubMedID: 26791865Scopus ID: 2-s2.0-84959509396OAI: oai:DiVA.org:kth-185057DiVA: diva2:920057
Note

QC 20160415

Available from: 2016-04-15 Created: 2016-04-11 Last updated: 2017-03-28Bibliographically approved
In thesis
1. Characterizing Chromatography Media: NMR-based Approaches
Open this publication in new window or tab >>Characterizing Chromatography Media: NMR-based Approaches
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
chromatography, nuclear magnetic resonance, NMR, self-diffusion, magic-angle spinning, quantitative, distribution coefficient, pore size
National Category
Physical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-204572 (URN)978-91-7729-334-7 (ISBN)
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

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