Načrtovanje in razvoj peptidnih ligandov za afinitetno čiščenje protiteles

Kruljec, Nika (avtor), Bratkovič, Tomaž (mentor)

Monoklonska protitelesa spadajo med najpomembnejše biološke učinkovine s širokim razponom terapevtskih indikacij. Prvi korak v izolaciji in čiščenju monoklonskih protiteles je njihovo zajetje iz procesne tekočine z afinitetno kromatografijo. V ta namen najpogosteje uporabljamo bakterijske imunoglobulin-vezavne proteine (npr. stafilokokni protein A, SpA). Slednji imajo številne slabe lastnosti, zaradi česar obstaja vse večja težnja po razvoju novih afinitetnih ligandov. Eno izmed perspektivnejših skupin alternativnih ligandov predstavljajo peptidi. V okviru doktorskega dela smo s presejanjem kombinatoričnih bakteriofagnih predstavitvenih knjižnic peptidov identificirali skupino peptidnih ligandov z afiniteto do fragmenta Fc imunoglobulinov G. Med petimi peptidnimi vezalci z dvema različnima aminokislinskima motivoma se je z najboljšimi vezavnimi lastnostmi izkazal peptidni ligand 19Fc. Na osnovi kompeticije s SpA za vezavo na regijo Fc predvidevamo, da se vezavno mesto peptidnega liganda nahaja na stiku domen CH2 in CH3. Z namenom določitve minimalnega motiva in ključnih aminokislinskih ostankov peptida 19Fc za vezavo na fragment Fc smo skrajšali peptid na N- in/ali C-koncu ter izvedli alaninsko rešetanje. Kot rezultat smo dobili minimalizirano različico peptidnega liganda, poimenovano min19Fc. Interakcijo med sinteznim biotiniliranim peptidom b-min19Fc ter humanim fragmentom Fc smo potrdili s pomočjo površinske plazmonske resonance (SPR). V nadaljevanju smo s ciljem povečanja topnosti ter morebitnega povečanja občutljivosti interakcije min19Fc-regija Fc na spremembe vrednosti pH v peptidu min19Fc zamenjali za vezavo neesencialen aminokislinski ostanek z nabitimi aminokislinami (glutamat, aspartat in lizin). Mutantam smo ovrednotili afiniteto vezave do različnih podrazredov humanih IgG ter določenih terapevtskih monoklonskih protiteles. Nadalje smo pripravili dve skupini peptidnih mutant z zamenjavami izbranih aminokislinskih ostankov s sorodnimi ter s slednjimi skušali pridobiti boljši vpogled v odnos med strukturo in vezavnimi lastnosti peptidov. Interakcijo izbranega peptidnega afinitetnega liganda min19Fc ter izboljšane mutante min19Fc Q6D do humanih IgG smo ovrednotili tudi z mikrotermoforezo (MST). Sposobnost izolacije in čiščenja protiteles s sinteznimi peptidi smo ocenili s testi »pull down«. Tako smo simulirali pogoje, podobne tistim, ki so jim protitelesa izpostavljena pri potovanju skozi afinitetno kromatografsko kolono. Končni cilj doktorskega dela je bila priprava afinitetne kromatografske kolone z izbranim peptidnim ligandom, min19Fc Q6D. Sintezni peptid smo prek kratkega distančnika vezali na sefarozni matriks, aktiviran s cianogen bromidom. Tako pripravljena kolona je izkazovala dinamično vezavno kapaciteto 11 mg IgG/mL afinitetnega nosilca. Iz kompleksnih zmesi, kot sta celično gojišče in humani serum, smo izolirali hIgG z ocenjeno čistoto, ki presega 95 %, ter s tem potrdili visoko specifičnost afinitetne kolone. Izpostavili smo jo več kot petindvajsetim poskusom izolacije hIgG in pri tem nismo zaznali znatnega zmanjšanja funkcionalnosti. Med preizkušenimi pogoji zaprtega čiščenja kromatografske kolone smo uporabili tudi raztopino 6 M gvanidinijevega klorida, 30 % izopropanol ter 1 M NaOH, kar ni bistveno vplivalo na kapaciteto afinitetne kolone, s čimer smo potrdili stabilnost razvite afinitetne kolone. Učinkovito elucijo IgG iz kolone smo dosegli z glicinskim pufrom s pH 2,5.

• Jezik: Slovenski jezik
• Ključne besede: afinitetna kromatografija, izolacija in čiščenje protiteles, peptidi, predstavitev na bakteriofagu
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2019
• PID: 20.500.12556/RUL-107353
• COBISS.SI-ID: 3972116
• Datum objave v RUL: 03.04.2019

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design and development of peptide ligands for antibody affinity purification

Izvleček

Monoclonal antibodies (mAb) have emerged as dominant biopharmaceuticals for treatment of various diseases. The initial step of therapeutic antibody downstream processing is antibody capture which typically relies on affinity chromatography. The most commonly used affinity matrices are based on bacterial immunoglobulin binding proteins such as staphylococcal protein A (SpA). However, such affinity ligands suffer from several drawbacks. Therefore, there is pressing need for new alternative affinity ligands. One of the most promising groups of alternative affinity ligands are short peptides. In this work, we have identified a new group of peptide affinity ligands from screening commercial phage display random peptide libraries. Among five peptide ligands with two different amino acid consensus motifs, the clone 19Fc was chosen for further characterization. Phage clone displaying the lead peptide 19Fc competed with immunoglobulin-binding staphylococcal protein A (SpA) for hFc binding, which indicates that the 19Fc peptide’s binding site at least partially overlaps with that of SpA (located at the CH2 and CH3 domain interface). Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide, termed min19Fc, for Fc binding. The interaction of the human Fc fragment with synthetic biotinylated peptide b-min19Fc was confirmed by surface plasmon resonance. With the goal of augmenting peptide’s solubility and possibly potentiating the pH-dependence of its interaction with the Fc fragment, we focused on modifying position 6 occupied by glutamine in the parental peptide min19Fc. Gln6 was substituted with charged residues (aspartate, glutamate, or lysine) yielding peptides min19Fc Q6D, min19Fc Q6E, and min19Fc Q6K, respectively. We analyzed the binding properties of modified peptides and compared them with the parental peptide min19Fc. In order to gain a better insight into the relationship between the structure and binding properties of the peptides, we have prepared two sets of peptide mutants with structurally similar amino acid substitutions at chosen positions. We used microscale thermophoresis (MST) method to quantitate the binding affinity of peptides min19Fc and min19Fc Q6D to human IgG pool. Pull-down assays with synthetic peptides were undertaken for simulation of column conditions to confirm that their interactions with IgGs are strong enough to support antibody isolation from complex mixtures, as well as to assess the peptides’ specificity. The ultimate goal of this PhD thesis was the construction of affinity chromatographic column based on optimized peptide affinity ligand min19Fc Q6D. Peptide min19Fc Q6D with the amidated GGDDK-NH2 C-terminal extension was coupled to a CNBr-activated Sepharose matrix. The dynamic binding capacity of the affinity column was estimated at 11.0 ± 1.5 mg IgG/mL affinity matrix. We subjected the column to human IgG purification from complex protein mixtures, consistently attaining antibodies with purities exceeding 95%, thus confirming high specifity for human Fc fragment. We observed no significant reduction of column's functional performance over more than 25 chromatographic runs. We have tested several cleaning in place (CIP) conditions, washing the column with 6 M guanidine chloride, 30% isopropanol, and 1 M NaOH, and observed no significant differences in DBC values, thus confirming the stability of the developed affinity column. Efficient elution from the column was achieved with glycine buffer of pH 2.5.

• Ključne besede: affinity chromatography, isolation and purification of antibodies, peptides, phage display