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Priprava in vrednotenje afinitetne kromatografske kolone na osnovi peptidnega liganda za izolacijo imunoglobulinov
ID Strašek, Maša (Author), ID Bratkovič, Tomaž (Mentor) More about this mentor... This link opens in a new window, ID Černigoj, Urh (Comentor)

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Abstract
Monoklonska protitelesa predstavljajo eno izmed najpomembnejših in vsestranskih skupin bioloških učinkovin. Za izolacijo in čiščenje terapevtskih protiteles uporabljamo pretežno afinitetno kromatografijo, ki temelji na bakterijskih imunoglobulin-vezavnih proteinih (npr. stafilokoknem proteinu A in streptokoknem proteinu G), a ima le-ta številne pomanjkljivosti. Cilj dela je bil pripraviti in ovrednotiti afinitetno monolitno kromatografsko kolono na osnovi peptidnega liganda za čiščenje in izolacijo imunoglobulinov G (IgG). Pripravili smo afinitetni koloni s stacionarno fazo iz polimetakrilatnega monolita in premrežene agaroze. Na funkcionalizirano površino nosilcev smo prek distančnikov tris(2-aminoetil)amina in bromoacetata imobilizirali peptidni ligand, označen s C-končnim cisteinskim ostankom. S sistemom HPLC smo analizirali dinamično vezavno kapaciteto IgG pripravljenih kromatografskih kolon. Pri agarozni peptidni koloni smo potrdili vezavo IgG, pri monolitni peptidni koloni pa do vezave ni prišlo. Na enako funkcionaliziran monolitni nosilec smo imobilizirali še druge modelne ligande, kot so oligonukleotidi (prek aminske ali tiolne skupine) ter protein A (prek cisteinskega ostanka). Tako pripravljene kromatografske kolone smo primerjali z monolitnimi kolonami z enakim ligandom, a z drugačno funkcionalizacijo. Z določanjem dinamične vezavne kapacitete smo potrdili delovanje vseh. Sklepamo, da hidrofobna narava peptidnega liganda IgG ni kompatibilna s prav tako relativno hidrofobnim polimetakrilatnim monolitnim nosilcem. Domnevamo, da zaradi hidrofobnih interakcij med obema peptidni ligand ni dostopen za vezavo protiteles.

Language:Slovenian
Keywords:afinitetna kromatografija, monolit, CIM, peptidni ligand, imunoglobulini
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2023
PID:20.500.12556/RUL-146490 This link opens in a new window
COBISS.SI-ID:154999299 This link opens in a new window
Publication date in RUL:03.06.2023
Views:789
Downloads:119
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Secondary language

Language:English
Title:Preparation and evaluation of an affinity chromatographic column based on a peptide ligand for immunoglobulin isolation
Abstract:
Monoclonal antibodies constitute one of the most important and versatile groups of biological drugs. Affinity chromatography based on bacterial immunoglobulin-binding proteins (e.g. staphylococcal protein A and streptococcal protein G) is typically used for isolation and purification of therapeutic antibodies, but it has many shortcomings. The aim of this work was to construct and evaluate an affinity monolithic chromatographic column based on a peptide ligand for the purification and isolation of immunoglobulin G (IgG). Affinity columns with polymethacrylate monolith and cross-linked agarose stationary phases were prepared. A peptide ligand labeled with a C-terminal cysteine residue was coupled to the functionalized supports via tris(2-aminoethyl)amine and bromoacetate spacers. The IgG dynamic binding capacity of the chromatographic columns was analyzed using an HPLC system. IgG binding was confirmed for the peptide: agarose matrix, but not for the peptide: monolith matrix. Using the same coupling chemistry, we prepared affinity matrices functionalized with different model ligands, such as oligonucleotides (via amino and sulfhidryl groups) and protein A (via a cysteine residue), and compared them to those carrying the same ligands but immobilized in a different way. All the affinity matrices were functional judging from the determined dynamic binding capacity. We conclude that the hydrophobic nature of the peptide IgG ligand is not compatible with the relatively hydrophobic polymethacrylate monolithic support. We assume that due to hydrophobic interactions between the two, the peptide is not accessible for antibody binding.

Keywords:affinity chromatography, monolith, CIM, peptide ligand, immunoglobulins

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