Sønderby, Pernille

Dannelse af makromolekylære komplekser og proteiners opførsel i vandige opløsninger er et resultat af protein-protein interaktion (PPI) og den kemiske natur af opløsningens komponenter. PPI kan deles i to brede kategorier som dækker over attraktive og frastødende kræfter. Fra et formulerings perspektiv er den rette interaktion kritisk for den langsigtede stabilitet af et lægemiddel. Protein kompleksdannelse er vigtig for forlængelse af halveringstid in vivo og er essentiel for cellulær kommunikation såsom induktion af insulinresponset.Denne afhandling fokuserer på human serum albumin (HSA) og brugen af småvinkel røntgen spredning (SAXS) til at studerer proteiner under forskellige opløsningsbetingelser. HSA benyttes på mange måder i den farmaceutiske industri, såsom i formuleringen af andre proteiner og navnligt for halveringstids-forlængelse af peptider, hvor den lange halveringstid for HSA på op til 21 dage bliver udnyttet.Afhandlingen er delt i fire hovedafsnit: 1) Selv-interaktion af HSA i farmaceutisk relevante systemer; 2) Liraglutid oligomeren og dennes interaktion med HSA; 3) Konjugering af GLP-1 til HSA og sidst men ikke mindst 4) Et benchmark studie af implementering af SAXS i RosettaDock.HSA benyttes som stabilisator i formuleringer, hvor det er kendt at den reducerer adsorption til luft/væske og væske/beholder grænseflader og hermed reducerer aggregerings tilbøjeligheden for mål proteinet. Der er desværre ikke megen information tilgængelig om opførelsen af HSA i mere tætpakkede systemer, som ved formuleringen af høj-koncentrations protein opløsninger. Vi har benyttet SAXS til at belyse selv-interaktionen af HSA i farmaceutisk relevante systemer i protein koncentrationer op til 150 mg/mL. Det ses at når HSA er stabiliseret af oktanoat, er interaktionen domineret af frastødende kræfter, mens affedtet HSA viser attraktion ved lav protein koncentration. En tonicitet svarende til isotonicitet, er observeret til at koincidere med maksimum screening af protein molekyler i vandig opløsning for alle systemer, mens dette ikke er tilfældet som en funktion af ionstyrke. Dette gør tonicitet til en vigtig parameter for protein interaktion i de undersøgte systemer. Trehalose ser ud til potentielt at screene tilsat NaCl, hvilket føre os til at tænke at trehalose på en eller anden måde bestemmer rækkevidden for interaktionen for proteiner i opløsning. Til sidst foreslås det at den stabiliserende effekt af HSA ved højere protein koncentration, kan opstå ved dannelsen af et netværk af frastødende HSA molekyler, som derved screener interaktionen mellem andre proteiner hvorved aggregerings tendensen vil formindskes.Glukagonlignende peptid-1 (GLP-1) er et inkretin hormon som benyttes i behandlingen af diabetes mellitus type 2. Da GLP-1 har en begrænset halveringstid på ca. 3 minutter, har Novo Nordisk A/S udviklet en acyleret analog, liraglutid, som på grund af multimerisering og en tilsyneladende interaktion med HSA in-vivo, gør en-gang-daglig administration mulig. Oligomerisering og interaktion med HSA var undersøgt ved hjælp as SAXS og statisk lys spredning (SLS). Liraglutid oligomeren viste sig at bestå af ca. 7 monomerer (heptamer), men grundet usikkerheder blev de eksperimentelle studier komplimenteret af en serie af molekyl-dynamisk (MD) simuleringer. Resultaterne fra MD blev sammenlignet med SAXS data og den kombinerede tilgang viste at heptameren var den mest sandsynlige multimer. Studierne af liraglutid med HSA i både en affedtet og stabiliseret form, kunne bekræfte eksistensen af et hetero kompleks men ved meget lav koncentration. Dette kan være et resultatet af en svag binding af liraglutid til HSA eller af ikke optimale pufferbetingelser.En anden tilgang til halveringstids-forlængelse er konjugering af molekyler til HSA. I denne del af afhandlingen er ny-skabte GLP-1-albumin konjugater udviklet af Albumedix A/S, undersøgt ved en kombineret tilgang af farmakokinetik og struktur analyse ved hjælp af SAXS. GLP-1 var konjugeret til Cys34 af rekombinant HSA (rHSA) og to HSA varianter med højere (HB) og lavere (NB) bindingsevne til neonatal Fc receptoren (FcRn). Affinitets studier viste at konjugeringen havde begrænset effekt på bindingsevnen af konjugaterne til FcRn sammenlignet med de respektive rHSA varianter. En øget in-vivo halveringstid af konjugaterne var observeret i NMRI WT mus sammenlignet med GLP-1, hvor HB varianten viste en omkring 300 gange længere halveringstid, mens potensen af GLP-1-albumin var nedsat 3-4 størrelsesordner sammenlignet med GLP-1. Strukturen af rHSA og konjugaterne i vandig opløsning indikere at den konjugaterde GLP-1 er fleksibel og peger væk fra overfladen af rHSA. Lav-resolutions strukturen fra SAXS kombineret med høj-resolutions information fra røntgen krystallografi, kan forklare de farmakokinetiske resultater, da begrænset interferens er observeret mellem FcRn og konjugaterne, mens sterisk hindring fra albumin kan forklare den nedsatte potens af konjugaterne.Struktur bestemmelse af makromolekylære komplekser, ved hjælp af traditionelle tilgange såsom røntgen krystallografi og NMR, kan være udfordrende. En alternativ tilgang til information om sådanne komplekser er brugen af eksperimentel viden i kombination med in-silico modellering. Det sidste kapitel af afhandlingen handler om en benchmarking af protein docking programmet, RosettaDock, kombineret med SAXS. RosettaDock protokollen består af to trin, hvor det første trin imiterer det indledende møde mellem de to protein partner. Under det næste trin bliver positionen af den mindre protein partner og sidekæde positioner mellem de to proteiner, optimeret. Vi benytter SAXS under det første trin (SAXSconstrain) for at begrænse det søgerum der er tilgængeligt for RosettaDock. Da det er en ”rigid body” tilgang vil større konformationelle ændringer, enten mellem de to proteiner eller på overfladen, begrænse metoden da det kan resultere i en overordnet form som ikke svarer overens med SAXS resultaterne. 38 strukturer fra Benchmark 4.0 med forskellige sværhedsgrader blev undersøgt. Generelt indikerer vores resultater at implantationen af SAXSconstrain begrænser søgerummet og øger chancen for at finde et kompleks af næsten oprindelig struktur. I et større perspektiv ligger styrken af RosettaDockSAXS i kombinationen af lav-opløsnings information fra SAXS og protein-protein interaktions energierne fra RosettaDock. Dette giver en mulighed for højere-opløsnings information om grænsefladen mellem to proteiner og åbner muligheden for mere nøjagtig forudsigelse af ukendte protein-protein komplekser.The formation of macromolecular complexes and the solution behaviour of proteins are results of protein-protein interactions (PPI) and the nature of the solution components. PPI’s can be divided into two broad categories of attractive and repulsive forces. From a formulation perspective, proper interaction is critical for the long term stability of a pharmaceutical. Protein complex formation is important for extended half-life in vivo and is essential to cellular communication such as the induction of the insulin response.This thesis focuses on human serum albumin (HSA) as a central player and the ability of small-angle X-ray scattering (SAXS) to study proteins under diverse solution conditions. HSA is utilized in many ways in the pharmaceutical industry such as the formulations of other proteins and in particular for half-life extension of peptides, where the long half-life of up to 21 days of HSA is deployed.The thesis is divided into four parts: 1) Self-interaction of HSA in pharmaceutical relevant systems; 2) The liraglutide oligomer and interaction with HSA; 3) Conjugation of GLP-1 to HSA and last but not least 4) A benchmark study of the implementation of SAXS in RosettaDock.HSA is used as a stabilizer in formulation where it is known to decrease adsorption to the air/liquid and liquid/container interfaces, thereby reducing the aggregation propensity of the target protein. However, with high concentration liquid formulations coming into focus limited information is available on the behaviour of HSA in these crowded environments. We have applied SAXS in order to shed light on the self-interaction of HSA in pharmaceutical relevant buffer systems on protein concentrations up to 150 mg/ml. HSA stabilized by octanoate is observed to interact by purely repulsive forces in the investigated concentration ranges while defatted HSA show attractive forces at low protein concentration. The tonicity corresponding to isotonicity, is observed to coincide with the maximum screening observed in all systems while this is not observed as a function of ionic strength, making tonicity an important parameter for protein interaction in the investigated systems. Trehalose is seen to provide a screening effect of added NaCl leading us to think that trehalose somehow determines the range of interaction of the proteins in solution. Finally it is proposed that the stabilizing effect of HSA could be mediated by a repulsive network of HSA molecules screening the interaction of other proteins, hereby decreasing the aggregation propensity.Glucagon-like peptide 1 (GLP-1) is an incretin hormone used in the treatment of diabetes mellitus type 2. Due to its very limited half-life of approximately 3 minutes, Novo Nordisk A/S developed an acylated analogue, liraglutide, which due to multimerization and probable interaction with HSA in-vivo, makes it applicable for once daily administration. The oligomerization and the interaction with HSA were investigated by SAXS and static light scattering (SLS). The oligomeric state of liraglutide was approximately heptameric but due to uncertainties, the experimental studies were complimented by a series of molecular dynamic (MD) simulations. The results from MD where compared to the experimental SAXS data and the combined effort show that the liraglutide heptamer was the most probable multimer. The studies of liraglutide with HSA both in a defatted form and a stabilized form, were capable of confirming the presence of a hetero complex though at very low concentration. This could be a result of very transient interaction or the consequence of non-optimal buffer conditions.Another approach to half-life extension is conjugation of molecules to HSA. In this part of the thesis, novel GLP-1-albumin conjugates developed by Albumedix A/S where examined by a combined approach of pharmacokinetic studies and solution structure determination with SAXS. GLP-1 was conjugated to Cys34 of recombinant HSA (rHSA) and two rHSA variants with lower (NB) and higher binding (HB) affinity to the neonatal Fc receptor (FcRn). Binding kinetics showed that the conjugation had limited effect on the binding properties of the conjugates to FcRn compared to the respective rHSA variants. Increased in-vivo half-life of the conjugates was observed in NMRI WT mice compared to GLP-1, with the HB-variant displaying ~300 times longer half-life, while the potency of GLP-1-albumin was decreased by 3-4 orders of magnitude compared to GLP-1. The solution structure of the rHSA variants and the conjugates indicate a flexible nature of the conjugate, with the GLP-1 pointing away from the surface of rHSA. The low resolution structure from SAXS combined with high resolution structural information from X-ray crystallography, explains the pharmacokinetics results, as limited interference is seen between FcRn and the conjugate, while albumin steric hindrance explains the decreased potency of the conjugates.Structure determination of macromolecular complexes can be challenging by traditional approaches such as X-ray crystallography and NMR. An alternative approach is the use of experimental knowledge in combination with in-silico modelling to gain knowledge about macromolecular complexes. The final part of the thesis regards the benchmarking of the protein docking tool, RosettaDock, in combination with SAXS. The RosettaDock protocol is a two-step process involving a low resolution rigid body protocol, set up to imitate the initial protein-protein encounter, followed by a high resolution protocol where the position of the proteins and amino acid side chains are optimized. We applied SAXS as constrain (SAXSconstrain) after the low resolution step to filter out complexes with overall shapes which would not match the SAXS data. Since it is a rigid body approach larger conformational changes in any part of the complex provides a limit to the method either from interface non-complementarity or a resulting shape which deviates from the SAXS data. 38 structures from Benchmark 4.0 of different difficulty levels were investigated using this approach. In general our result indicate that implementation of SAXSconstrain reduces the sampling space and increases the probability of finding a near-native structure. In a wider perspective, the strength of RosettaDockSAXS lies in the combination of low-resolution structural information from SAXS experiments and the protein-protein interaction energies obtained from RosettaDock which provides means to gain insight to higher resolution information about the interface between two protein partners. This allows for the prediction of unknown three-dimensional atomic structures of protein-protein complexes

Online Research Database In Technology

Solution behaviour of Human Serum Albumin and GLP-1variants

http://orbit.dtu.dk/ws/files/127486121/Pernille_S_nderby_phd._afhandling.pdf

Solution behaviour of Human Serum Albumin and GLP-1variants

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