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Tescari, Simone (2017) Production and characterization of therapeutic proteins/peptides: Human Recombinant FSH-beta subunit expressed in Plant Cells and Chemical Synthesis of Human Osteocalcin and Neuritogenic Peptides. [Ph.D. thesis]

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Abstract (english)


Since 1980s proteins have emerged as a major new class of pharmaceuticals with over 350 marketed products that are mainly therapeutics with a small number of diagnostics and vaccines. In 2015 among the 10 best-selling drugs in the world, 7 are recombinant proteins or monoclonal antibodies. There is still considerable confusion about what therapeutic proteins are. Regulatory authorities (U S Food and Drug Administration and European Medicines Agency) provided different definitions describing them as Biological medicinal products, Biopharmaceuticals, Biological or Biologic depending on production system, biological source and pharmaceutical category. The European Medicines Agency (EMA) provided an actual definition describing biological medicinal products as “a protein or nucleic acid–based pharmaceutical substance used for therapeutic or in vivo diagnostic purposes, which is produced by means other than direct extraction from a native (non-engineered) biological source”. This definition suggests that even small polypeptides and proteins obtained by chemical synthesis can be considered as therapeutic proteins.
Nowadays, most of approved therapeutic proteins are produced by DNA recombinant technologies. Recombinant proteins are mainly restricted to mammalian cells (CHO) as bioreactors which carry out post-translational modifications that significantly enhance the protein bioactivity. Recently, plants are emerging as an attractive alternative to conventional expression systems, due to its practical, economic and safety advantages, correct folding and similar glycosylation pattern like eukaryotes. In 2013 U S Food and Drug Administration approved the first plant-made pharmaceutical Elelyso® (Protalix and Pfizer) to treat Gaucher’s Disease.
In this scenario, Active Botanicals Research (Brendola, VI, Italy) proposes suspension cell lines of N. benthamiana as alternative expression platform for recombinant protein and in particular for recombinant human follicle stimulating hormone beta subunit. Follicle Stimulating Hormone (FSH) is a gonadotropin that stimulates steroidogenesis and gametogenesis in the gonads in order to support and regulate the ovarian follicular maturation in women and sperm production in men. FSH is clinically used for controlled ovarian stimulation in women treated with assisted reproductive technologies, the treatment of anovulatory infertility in women and hypogonadotropic hypogonadism in men. FSH is most often administered in one of two forms: recombinant FSH expressed in CHO system (Gonal-F® (Merck Serono) and Puregon® (Merck Sharp and Dohme)) or highly purified human menopausal gonadotropin (Menopur® (Ferring) and Merional® (Pharmasure)). The first chapter of this thesis focuses on extraction, purification and characterization of recombinant human follicle stimulating hormone beta subunit expressed in suspension cell lines of N. benthamiana. Extraction is based on KDEL-strategy, a retention signal which locks the protein in the endoplasmatic reticulum preventing the release into the cytoplasm. Purified recombinant human Follicle Stimulating Hormone beta subunit (rhFSHβ) was obtained with three consecutive chromatographic steps: IMAC chromatography, size-exclusion chromatography and ion-exchange chromatography.
Two purified isoforms of recombinant protein were chemically characterized by enzymatic deglycosylation with PNGase F and tryptic digestion, covering the total amino acid sequence while the assignment of disulphide bridges pattern (six internal disulphide bridges) confirmed the correct folding of protein.
It is known that FSH evokes the physiological response as a heterodimer. In all glycoproteins the common alpha subunit is non-covalently associated whit the beta subunit, which is structurally unique in its peptide sequence to each member of the family. However, some studies suggest a possible biological activity about the single beta subunit and different FSH beta subunit preparations are on market declaring its biological activity (FSHβ subunit ab191730 Abcam).
Purified rhFSHβfrom ABResearch was tested on isolated Sertoli cells from pubertal porcine, unfortunately, preliminary data suggested no biological activity of the recombinant monomer. Waiting for recombinant human FSH alpha subunit from competent cell lines of N. Benthamiana, the biological activity of reconstituted heterodimer (rhFSHβfrom ABResearch + Native Human Chorionic Gonadotropin) was evaluated and compared with commercial FSH Gonal-F®. Data showed equal biochemical responses suggesting the great potential of the new expression system for active recombinant therapeutic proteins.
The process has been improved to obtain 4.5/5 mg of purified rhFSHβ for kg of cells, exceeding abundantly average yields in plant expression system.
In the second chapter of this work, chemical synthesis of human osteocalcin (OC) 1-49 by solid-phase synthesis (SPPS) with Fmoc strategy is described. Human OC is a small protein of 49 amino acid residues mainly produced and secreted by osteoblasts, it represents one of the most abundant (10-20%) non-collagenous proteins in the bone tissue of vertebrates with a highly conserved primary structure. It has long been known that OC acquires high affinity for calcium ions through the post-translational modification of glutamate residues by carboxylation with a vitamin-K-dependent γ-glutamyl carboxylase (GGCX). Carboxylated residues, known as Gla residues, lead to a conformational change, stabilize the alpha-helical structure and confer a greater affinity for Ca2+ and hydroxyapatite. OC is involved in different diagnostic fields due to its crucial roles in several physiological processes including remodeling of bone tissue, insulin production and regulation, testosterone secretion from testes and regulation of neurotransmitter levels in the brain. Recently osteocalcin has been proposed as potential therapeutic protein in regulation of androgen activity in a non-steroidal manner.
Nowadays purified human OC 1-49 is obtained by extraction from human bones or solid-phase synthesis with the Boc strategy. Due to limited supply of human osteocalcin from bone, the solid-phase synthesis represents the most accessible strategy.
In this study, SPPS Fmoc chemistry was proposed as alternative strategy due to the greater yields of synthesized peptides, minor side reactions during cleavage and high safety compared to Boc approach. Chemical synthesis was optimized getting final yield of 80/85% exceeding average yields of traditional chemical synthesis. The purified protein is subjected to the disulfide bond-forming reaction and subsequently it was chemically characterized by RP-HPLC, mass spectrometry analysis and enzymatic digestion. Conformational characterization and study of binding with Ca2+ were performed by spectroscopic measurements, hydrogen/deuterium exchange and ITC titration analysis comparing the new synthesized human osteocalcin with the commercial one (Bachem).
Peptides as therapeutics are the focus of the third chapter.
Peptides perform crucial roles in human physiology as growth factors, hormones, ion channel ligands, neurotransmitters and they are recognized for being highly selective and efficacious signaling molecules with attractive pharmacological profile. Peptides represent the new attractive perspective for therapeutics due to high safety, tolerability and efficacy properties. Lower production complexity and lower cost also increase interest in peptide drugs research and development compared with protein therapeutics.
Nowadays synthesized peptides are used in regenerative medicine due to their potential as guidance cues for neurite elongation and thus to activate intracellular pathways leading to cell differentiation.
Ten novel peptides derived from cell adhesion molecules and extracellular matrix proteins families (CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, LINGO2, Contactin 1, 2 and 5) are designed to mimic guidance cues from the neural environment.
Peptides are synthesized with SPPS Fmoc strategy and characterized by RP-HPLC, mass spectrometry and circular dichroism analyses. According to previous experiments with L1-A and LINGO1-A, all synthetic peptides are tested on human neuroblastoma cell lines to evaluate their effect on neuronal differentiation and especially in neurite outgrowth and elongation.
Preliminary data suggest a prototype for the development of implants for long-term neuronal growth and differentiation.

Abstract (italian)

Dagli anni 80, le proteine terapeutiche sono emerse come la classe di farmaci più promettenti con più di 350 prodotti in commercio tra terapeutici, diagnostici e vaccini. Nel 2015 tra i 10 farmaci più venduti al mondo, 7 sono proteine ricombinanti od anticorpi monoclonali.
C’è ancora confusione relativamente al concetto di proteina terapeutica. Le autorità regolatorie come FDA ed EMA hanno dato diverse definizioni sulla base dei sistemi di produzione, delle fonti biologiche e della categoria farmaceutica di appartenenza.
L’Agenzia Europea dei Medicinali (EMA) descrive i prodotti medicinali biologici come sostanze farmaceutiche di natura proteica (o acidi nucleici) utilizzate in vivo per scopi terapeutici o diagnostici, le quali sono prodotte con metodologie diverse dalla diretta estrazione da fonti biologiche native (non ingegnerizzate geneticamente). Questa definizione suggerisce che anche le piccole proteine e peptidi prodotti chimicamente siano considerate proteine terapeutiche.
Oggigiorno, la maggior parte delle proteine approvate per scopi terapeutici sono prodotte con la tecnologia del DNA ricombinante. Le cellule ovariche di criceto cinese (CHO) sono considerate i bioreattori per eccellenza poiché sono in grado di apportare le modifiche post-traduzionali adatte e necessarie per una corretta attività biologica. Recentemente le piante sono emerse come un’attraente alternativa ai normali sistemi di espressione grazie ai vantaggi di tipo economico, di sicurezza, del corretto folding della proteina e del pattern di glicosilazione simile a quello umano. Nel 2013 l’autorità regolatoria americana ha approvato il primo farmaco proteico prodotto interamente in pianta contro la malattia di Gaucher.
In questo scenario, Active Botanicals Research (Brendola, VI, Italia), ha proposto linee cellulari in sospensione da N. benthamiana come piattaforma alternativa per l’espressione di proteine ricombinanti ed in particolare per la sub unità beta dell’ ormone follicolo stimolante (FSH). L’ormone follicolo stimolante è una gonadotropina che stimola la produzione di steroidi e dei gameti nelle gonadi per supportare e regolare la maturazione follicolare nelle donne e degli spermatozoi nei maschi. FSH è utilizzato clinicamente per controllare la stimolazione ovarica nella fecondazione assistita, nei casi d’infertilità femminile e ipogonadismo maschile.
FSH è somministrato in due forme: la proteina ricombinante espressa in CHO (Gonal-F® (Merck Serono) and Puregon® (Merck Sharp and Dohme)) o purificata dall’urina di donne in menopausa (Menopur® (Ferring) and Merional® (Pharmasure)).
Il primo capitolo di questa tesi affronta il processo di estrazione, purificazione e caratterizzazione della sub unità beta dell’ormone follicolo stimolante espresso in cellule in sospensione di N. benthamiana. L’estrazione sfrutta il KDEL: un segnale di ritenzione che è aggiunto all’estremità C-terminale della proteina e che permette di bloccarla all’interno del reticolo endoplasmatico. Il prodotto purificato è stato ottenuto in seguito a tre step cromatografici consecutivi: cromatografia di affinità (IMAC), ad esclusione molecolare e in fine a scambio ionico.
Due isoforme della stessa proteina ricombinante sono state caratterizzate chimicamente mediante deglicosilazione enzimatica con PNGase F e digestione triptica in modo da coprire totalmente la sequenza aminoacidica e in secondo luogo assegnare tutti e sei i ponti disolfuro dimostrando il corretto folding della proteina.
E’ risaputo che l’ormone follicolo stimolante svolge la sua funzione come etero dimero. In tutte le glicoproteine, la comune subunità alpha è legata in modo non covalente alla subunità beta, la quale è unica nella sequenza e struttura per ciascun membro della famiglia. Tuttavia, alcuni studi suggeriscono una possibile attività biologica da parte della singola subunità beta paragonabile all’intero etero dimero. In commercio è presente una preparazione con dichiarata attività biologica (FSHβ subunit ab191730 Abcam).
La specie purificata da ABResearch è stata testata su cellule isolate del Sertoli da testicolo di maiale. I dati preliminari hanno dimostrato l’assenza di attività biologica del singolo monomero. Aspettando la produzione della subunità alpha, l’etero dimero tra la subunità beta di ABR e la subunità alpha commerciale è stato costituito e testato dimostrando un’attività paragonabile al prodotto commerciale.
I dati sperimentali sostengono quindi le potenzialità di questo nuovo sistema di espressione di proteine terapeutiche, ottenendo inoltre delle rese che superano notevolmente le rese medie ottenute dai classici sistemi vegetali (4.5/5 mg per Kg di cellule).
Nel secondo capitolo di questo lavoro è descritta la sintesi chimica di osteocalcina umana 1-49 (OC) mediante la sintesi di peptidi su fase solida con strategia Fmoc.
Osteocalcina umana è una piccola proteina di 49 amino acidi prodotta principalmente dagli osteoblasti e rappresenta una delle proteine più abbondanti nel tessuto osseo dei vertebrati con un altissimo grado di conservazione della struttura primaria. E’ risaputo che osteocalcina acquisisce la sua caratteristica affinità per gli ioni calcio in seguito a specifiche modifiche post traduzionali da parte dell’enzima vitamina K dipendente glutamil-carbossilasi a livello di specifici residui di acido glutammico. Il binding con gli ioni calcio stabilizza la struttura alpha elica della proteina.
OC è coinvolta in diversi campi diagnostici a causa del suo ruolo cruciale in diversi processi fisiologici che comprendono il rimodellamento osseo, produzione e regolazione di insulina, secrezione di testosterone a livello testicolare e regolazione di neuro trasmettitori. Recentemente è stata proposta come proteina capace di regolare l’attività androgena in modo non steroideo.
Oggigiorno OC è ottenuta mediante estrazione da ossa di defunti o mediante sintesi chimica su fase solida con strategia di Boc. In seguito alla scarsa disponibilità di tessuto osseo e agli ovvi problemi etici, la sintesi chimica rappresenta la principale via di approvvigionamento.
In questa tesi si propone una strategia alternativa (Fmoc chemistry) conosciuta per le maggiori rese di reazione, vantaggi economici, maggiore sicurezza e minore contributo di reazioni collaterali rispetto alla classica strategia di Boc.
La sintesi chimica è stata ottimizzata fino a ottenere una resa del 80/85% superando largamente le rese medie della classica sintesi chimica. La proteina purificata è stata soggetta al processo ossidativo per la costituzione del ponte disolfuro ed ottenere così la proteina nella forma nativa. In seguito alla caratterizzazione chimica mediante RP-HPLC e spettrometria di massa, OC è stata caratterizzata da un punto di vista conformazionale mediante tecniche spettroscopiche come dicroismo circolare nel lontano UV e fluorescenza. Studi di binding al calcio sono stati condotti mediante l’utilizzo di tecniche spettroscopiche paragonando OC sintetizzata con quella commerciale (Bachem).
Il terzo capitolo riguarda i peptidi come prodotti terapeutici.
I peptidi ricoprono ruoli fisiologici cruciali come fattori di crescita, ormoni, ligandi di canali ionici, neurotrasmettitori e sono riconosciuti per la loro alta selettività ed efficacia come segnali molecolari con un attraente profilo farmacologico. I peptidi rappresentano una nuova attraente prospettiva nel settore farmaceutico grazie al loro grado di sicurezza, tollerabilità ed efficacia. La minor complessità di produzione e prezzi notevolmente più contenuti ha attirato l’interesse della ricerca scientifica sui peptidi come potenziali farmaci rispetto alle proteine terapeutiche.
Oggigiorno i peptidi sintetizzati vengono utilizzati nella medicina rigenerativa grazie al loro potenziale nella trasmissione del segnale, elongazione di neuriti e differenziazione cellulare.
Dieci nuovi peptidi derivanti da molecole di adesione cellulare e proteine della matrice cellulare (CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, LINGO2, Contactin 1, 2 and 5) sono stati progettati per mimare i segnali guida nell’ambiente neurale. I peptidi sono stati sintetizzati su fase solida e purificati mediante RP-HPLC, analizzati mediante spettrometria di massa e dicroismo circolare. In accordo con i dati ottenuti con i peptidi L1-A e LINGO1-A, tutti e dieci i peptidi testati su linee cellulari di neuroblastoma umano hanno dimostrato di essere efficaci nella differenziazione neuronale ed in particolare nella crescita ed elongazione dei neuriti.
Questi dati preliminari suggeriscono un prototipo per lo sviluppo di impianti per la crescita e differenziazione neuronale a lungo termine.


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EPrint type:Ph.D. thesis
Tutor:De Filippis, Vincenzo
Ph.D. course:Ciclo 29 > Corsi 29 > SCIENZE MOLECOLARI
Data di deposito della tesi:31 January 2017
Anno di Pubblicazione:31 January 2017
Key Words:Therapeutic Proteins-Biopharmaceuticals- FSH-Osteocalcin-Biomimetic Peptides
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Area 03 - Scienze chimiche > CHIM/08 Chimica farmaceutica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze del Farmaco
Codice ID:10291
Depositato il:02 Nov 2017 15:51
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