Application of mixed mode chromatography for separation of biologically important molecules

Kristl, Anja (author), Pompe, Matevž (mentor)

The development of a chromatographic method often involves a trial-and-error approach until the set criteria are met. The advantage of such a tactic is the development of an adequate method in a relatively short period of time. However, the lack of a systematic approach may lead us in a local optimum and unaware of the critical parameters that affect separation. This severely hinders our ability to troubleshoot when the performance of the method falls out of the accepted range. The educational guesses of chromatography experts can solve many problems encountered when separating on a column with a single retention mode. Unfortunately, such knowledge is not sufficient to decipher the optimal path to improve separation on a mixed mode stationary phase. The presence of multiple retention mechanisms in combination with very complex molecules such as proteins and other biomolecules renders such predictions extremely difficult. In this case, a good optimization path is the Quality by Design approach. Therefore, the aim of this study is to understand the principles of the governing factors. Our work began with screening the most suitable stationary phase chemistry for the separation of seven insulin variants commonly used in the treatment of diabetes mellitus. Before optimizing the composition of the mobile phase and gradients of acetonitrile content, buffer concentration, and pH value, we focused on the often neglected effects of temperature and pressure on separation efficiency. These effects were studied separately on appropriate single mode columns, as the selected mixed mode column included a reversed-phase and anion exchange mechanism. The effect of temperature on the separation of insulin is opposite to that of small molecules on both columns up to 55 °C. At higher temperature, the separation of insulin on the anion exchange column shows a similar trend as before. On the reversed phase and temperatures above 55 °C, insulin retains like a small molecule. The effect of pressure was observed only on the reversed-phase and the mixed mode column. In these cases, the retention of insulins increased significantly even when the column inlet pressure was increased by 100 bar. The retention of small molecules was only slightly affected. This was not observed for separations on an anion exchange column due to the non-denaturing mobile phase and thus the stability of the insulin molecule. This pressure effect on an anion exchange column was further studied with a probe molecules (oligonucleotides of different lengths), larger proteins (BSA and thyroglobulin) and a plasmid DNA molecule. A significant increase in retention time was observed for isocratic and gradient separations, which was dependent on the size and flexibility of the molecules. To investigate the adsorption mechanism, these separations were described using stoichiometric displacement and linear gradient elution models. A pressure and ionic strength dependence of distribution constant was developed and derived to obtain partial molar volume changes. Analysis of the calculated parameters indicated a compression of the macromolecules towards the stationary phase upon adsorption. This enabled the molecules to have more interactions with the stationary phase. Finally, we conducted a systematic study of the influence of mobile phase composition on the separation efficiency of seven insulin variants and two excipients on a mixed mode column. In addition, an SPE purification procedure was developed to remove interferences present in the formulations. Two separation methods were developed, each suitable for the separation of nine molecules on HPLC systems with either binary or quaternary solvent delivery system. The methods enable the quantification of human insulin and the six most commonly used therapeutic analogues in formulations or pharmaceutical raw materials.

• Language: English
• Keywords: chromatography, mixed mode, high pressure, macromolecules, insulin
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-131580
• COBISS.SI-ID: 84714755
• Publication date in RUL: 29.09.2021

Secondary language

• Language: Slovenian
• Title: Uporaba kromatografije z mešanimi režimi za separacijo biološko pomembnih molekul

Abstract

Razvoj metode kromatografske metode pogosto vključuje pristop poskusov in napak, dokler ne dosežemo zastavljenih kriterijev. Prednost takšnega načina je razvoj ustrezne metode v sorazmerno kratkem času. Pomanjkanje sistematičnega pristopa pa nas lahko vodi v lokalni optimum s slabim poznavanjem kritičnih parametrov, ki vplivajo na ločevanje. To močno ovira našo zmožnost odpravljanja težav, kadar učinkovitost metode pade iz sprejemljivega obsega. Nasveti strokovnjakov na podlagi izkušenj iz kromatografije lahko rešijo številne težave, ki se pojavijo pri ločevanju na koloni z enim samim načinom zadrževanja. Na žalost takšno znanje ne zadošča za dešifriranje optimalne poti za pravilno ločitev na stacionarni fazi z mešanim režimom. Prisotnost več mehanizmov zadrževanja v kombinaciji z zelo zapletenimi molekulami, kot so beljakovine in druge biomolekule, onemogoča takšne napovedi. Za take primere lahko optimizacijo dosežemo z metodo vgrajene kakovosti (angl. Quality by Design). Zato je cilj te študije spoznati in razumeti vodilne dejavnike pri razvoju tovrstnih separacijskih metod. Naše delo se je začelo s pregledom najprimernejših kemijskih lastnosti stacionarnih faz za ločevanje sedmih variant inzulina, ki se pogosto uporabljajo pri zdravljenju diabetesa mellitusa. Pred optimizacijo sestave mobilne faze in gradientov vsebnosti acetonitrila, koncentracije pufra in pH smo se osredotočili na vpliv temperature in tlaka, ki sta pogosto zanemarjena dejavnika na učinkovitost ločevanja. Te učinke smo preučevali ločeno na ustreznih kolonah z enim mehanizmom zadrževanja, da smo izsledke lahko uporabili na koloni z anionsko izmenjevalnim in reverznofaznim mehanizmom ločevanja. Vpliv temperature na ločevanje inzulina je nasproten učinku majhnih molekul na obeh kolonah do 55 °C. Pri višji temperaturi poteka ločevanje inzulina na anionsko izmenjevalni koloni podobno kot prej, na reverznifazi pa se zadržuje kot majhna molekula. Vpliv tlaka smo opazili le na RP koloni in koloni z mešanimi režimi. V teh primerih se je zadrževanje inzulinov znatno povečalo, tudi ko se je tlak pri vstopu v koloni povečal za 100 barov. Na zadrževanje majhnih molekul je bil vpliv tlaka precej manjši. Tega niso opazili pri ločevanju na anionsko izmenjevalni koloni zaradi nedenaturirajoče mobilne faze in s tem stabilnosti molekule inzulina. Ta učinek tlaka na anionsko izmenjevalni koloni smo nadalje preučevali z modelnimi molekulami (oligonukleotidi različnih dolžin), večjimi beljakovinami (BSA in tiroglobulin) in molekulo plazmidne DNA. Opazili smo znatno povečanje zadrževalnega časa pri izokratskih in gradientnih ločbah, kar je bilo odvisno od velikosti in fleksibilnosti molekul. Za raziskovanje adsorpcijskega mehanizma smo ta ločevanja opisali z uporabo modelov stehiometrične izmenjave in linearne gradiente elucije. Razvili smo model odvisnost porazdelitvene konstante od tlaka in ionske jakosti, z odvajanjem tega pa smo izračunali spremembe parcialnega molskega volumna. Analiza izračunanih parametrov je pokazala kompresijo makromolekul proti stacionarni fazi ob adsorpciji, česar posledica je več interakcij s stacionarno fazo. Na koncu smo izvedli še sistematično študijo vpliva sestave mobilne faze na učinkovitost ločevanja sedmih variant inzulina in dveh pomožnih snovi na koloni z mešanimi režimi. Poleg tega smo razvili postopek čiščenja SPE za odstranjevanje motenj, prisotnih v formulacijah. Razvili smo dve metodi ločevanja, vsaka primerna za ločevanje devetih molekul na HPLC sistemih z binarnim ali kvartarnim sistemom za dovajanje topila. Metode omogočajo kvantificiranje človeškega inzulina in šestih najpogosteje uporabljenih terapevtskih analogov v formulacijah ali farmacevtskih surovinah.

• Keywords: kromatografija, mešani režimi, visok tlak, makromolekule, insulin