Mixed-mode chromatographic materials are more and more often used for the purification of bio-molecules, such as peptides and proteins. In many instances they in fact exhibit better selectivity values and therefore improve the purification efficiency compared to classical materials. In this thesis, five cation-exchange reversed-phase (CIEX-RP) mixed-mode containing different proportions of sulfonic groups, three anion-exchange reversed-phase (AIEX-RP) containing different amount of ammonium groups and a purely reversed-phase material (RP) were used to study the impact of charged ligands on the purification of peptides. The purification of two synthetic peptides from their crude mixture and the separation of insulin from desamido-insulin were used as case study. A model to describe bio-molecules retention in cation-exchange/reversed-phase (CIEX-RP) mixed-mode columns under diluted conditions has been developed. The model accounts for the effect of the salt and organic modifier concentration on the bio-molecule Henry coefficient through three parameters α, β and γ. According to the model, the α parameter is related to the adsorption strength and ligand density, β represents the number of organic modifier molecules necessary to displace one adsorbed biomolecule and γ represents the number of salt molecules necessary to desorb one biomolecule. The latter parameter is strictly related to the number of charges on the bio-molecule surface interacting with the ion-exchange ligands and it is shown experimentally that its value is very close to the biomolecule net charge. The model reliability has been validated by a large set of experimental data including retention times of two different peptides (a synthetic peptide and insulin) on five columns: a reversed-phase C8 column and four CIEX-RP columns with different percentages of sulfonic groups and various concentration values of the salt and organic modifier. The influence of the cation exchange groups amount has been evaluated. It has been found that the percentage of sulfonic groups on the surface strongly affects the peptides adsorption strength, and in particular, in the cases investigated, a CIEX ligand density around 0.04 μmol/m2 leads to optimal retention values. Moreover, the purification efficiency of the three peptides mixtures considered was evaluated on both the AIEX-RP and CIEX-RP materials. Improvement in the purification has been noticed for both synthetic peptides with the yield increasing with the percentage of quaternary ammonium ions on the surface of the columns, whereas the purification on CIEX-RP columns was found to be less efficient than on regular RP columns. The selectivity between insulin and desamido-insulin has been improved with AIEX-RP columns showing a rising value of the selectivity with the percentage of positive charges on the stationary phase, whereas the selectivity was found to be nearly one on CIEX-RP materials.
La cromatografia mixed-mode è usata sempre di piu’ per la purificazione di biomolecole, come peptidi e proteine. In molti casi infatti mostra migliori valori di selettività e di conseguenza migliora l’efficienza di purificazione in confronto con i metodi classici. In questa tesi, sono state utilizzate per studiare l’impatto dei leganti carichi sulla purificazione di peptidi cinque colonne mixed-mode a scambio cationico in fase inversa (CIEX-RP) con differenti percentuali di gruppi solfonici, tre colonne a scambio anionico in fase inversa con differenti percentuali di gruppi ammonici (AIEX-RP) e una colonna di cromatografia in fase inversa (RP) . La purificazione di due diversi peptidi e la separazione dell’insulina dalla desamido-insulina sono state utilizzate come casi studio. È stato sviluppato un modello per descrivere la ritenzione delle biomolecole in colonne CIEX-RP in condizioni diluite. Il modello tiene in considerazione gli effetti della concentrazione del sale e del modificatore organico sul coefficiente di Henry delle biomolecole attraverso tre parametri α, β e γ. Secondo il modello, il parametro α è legato alla forza di adsorbimento e alla densità dei leganti, β rappresenta il numero di molecole del modificatore organico necessarie per rimuovere una biomolecola adsorbita e γ rappresenta il numero di molecole di sale necessarie per far desorbire una molecola. L’ultimo parametro è strettamente legato al numero di cariche sulla superficie della biomolecola che interagiscono con i leganti a scambio ionico e si mostra sperimentalmente che questo valore e molto simile alla carica netta della biomolecola. La validità del modello è stata sostenuta da un grande numero di dati sperimentali dei tempi di ritenzione di due peptidi (un peptide sintetico e l’insulina) su cinque colonne: una colonna RP e quattro colonne CIEX-RP con differenti percentuali di gruppi solfonici cambiando le concentrazioni del sale e del modificatore organico. Valutando l’influenza dei gruppi a scambio cationico si nota che la percentuale di gruppi solfonici sulla superficie influisce molto sulla forza di adsorbimento dei peptidi, e in particolare, nei casi indagati, una densità di leganti di 0.04 μmol/m2 conduce a un tempo di ritenzione ottimale. Inoltre, l’efficienza di purificazione di tre peptidi è stata valutata sia su materiali AIEX-RP sia su materiali CIEX-RP. Miglioramenti nella purificazione sono stati notati per entrambi i peptidi sintetici con un aumento delle rese con la percentuale di cariche positive sulla superficie delle colonne, mentre la purificazione attraverso colonne CIEX-RP si è rilevata meno efficiente della purificazione attraverso colonne RP. La selettività tra l’insulina e la desamido-insulina è migliorata usando colonne AIEX-RP, mostrando dei valori crescenti di selettività con la percentuale di cariche positive sulla fase stazionaria.
Peptide purification by mixed mode chromatography
BERNARDI, SUSANNA
2011/2012
Abstract
Mixed-mode chromatographic materials are more and more often used for the purification of bio-molecules, such as peptides and proteins. In many instances they in fact exhibit better selectivity values and therefore improve the purification efficiency compared to classical materials. In this thesis, five cation-exchange reversed-phase (CIEX-RP) mixed-mode containing different proportions of sulfonic groups, three anion-exchange reversed-phase (AIEX-RP) containing different amount of ammonium groups and a purely reversed-phase material (RP) were used to study the impact of charged ligands on the purification of peptides. The purification of two synthetic peptides from their crude mixture and the separation of insulin from desamido-insulin were used as case study. A model to describe bio-molecules retention in cation-exchange/reversed-phase (CIEX-RP) mixed-mode columns under diluted conditions has been developed. The model accounts for the effect of the salt and organic modifier concentration on the bio-molecule Henry coefficient through three parameters α, β and γ. According to the model, the α parameter is related to the adsorption strength and ligand density, β represents the number of organic modifier molecules necessary to displace one adsorbed biomolecule and γ represents the number of salt molecules necessary to desorb one biomolecule. The latter parameter is strictly related to the number of charges on the bio-molecule surface interacting with the ion-exchange ligands and it is shown experimentally that its value is very close to the biomolecule net charge. The model reliability has been validated by a large set of experimental data including retention times of two different peptides (a synthetic peptide and insulin) on five columns: a reversed-phase C8 column and four CIEX-RP columns with different percentages of sulfonic groups and various concentration values of the salt and organic modifier. The influence of the cation exchange groups amount has been evaluated. It has been found that the percentage of sulfonic groups on the surface strongly affects the peptides adsorption strength, and in particular, in the cases investigated, a CIEX ligand density around 0.04 μmol/m2 leads to optimal retention values. Moreover, the purification efficiency of the three peptides mixtures considered was evaluated on both the AIEX-RP and CIEX-RP materials. Improvement in the purification has been noticed for both synthetic peptides with the yield increasing with the percentage of quaternary ammonium ions on the surface of the columns, whereas the purification on CIEX-RP columns was found to be less efficient than on regular RP columns. The selectivity between insulin and desamido-insulin has been improved with AIEX-RP columns showing a rising value of the selectivity with the percentage of positive charges on the stationary phase, whereas the selectivity was found to be nearly one on CIEX-RP materials.File | Dimensione | Formato | |
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https://hdl.handle.net/10589/45042