Development of the production process for a recombinant hormone and evaluation of its biological activity

• Main Author: Font Ingles, Albert
• Other Authors: Valero Barranco, Francisco
• Format: Doctoral Thesis
• Language: English
• Published: Universitat Autònoma de Barcelona 2012
• Subjects: Tecnologies; 579 - Microbiologia
• Online Access: http://hdl.handle.net/10803/123298

Durant el curs d’aquest treball, es va dur a terme l’expressió de l’hormona Pregnant Mare Serum Gonadotropin (PMSG), també coneguda com equine Chorionic Gonadotropin (eCG) en el llevat metilotròfic Pichia pastoris. La PMSG consta de dues subunitats que varen ser expressades sota control del promotor AOX1. També es va desenvolupar l’upstream i el downstream del procés de producció per la PMSG recombinant (rPMSG). En primer lloc es va desenvolupar un assaig in vitro amb cèl·lules de Leydig (MLTC-1) per tal de validar que la conformació de l’hormona recombinant fos la correcta. En segon lloc, es van construir dos vectors d’expressió amb la Human Serum Albumin (HSA) fusionada a l’extrem C-terminal i N-terminal de la subunitat beta de la PMSG. A més, aquests vectors també contenien una còpia del gen de la subunitat alfa sota el control d’un promotor AOX1 independent. Els 12 punts d’O-glicosilació presents en la regió del C-terminal peptide (CTP) de la subunitat beta van ser eliminats amb l’objectiu de reduir la capacitat immunogènica que tenen els glicans de les proteïnes expressades en llevat. Eliminant tots els punts d’O-glicosilació la proteïna hagués vist reduït dràsticament el seu temps de vida mitja en sang, per aquesta raó es va decidir fusionar-la a la HSA amb l’objectiu de restablir aquesta mancança. Els primers cultius a petita escala van deixar entreveure que la fusió gènica de la HSA a l’extrem N-terminal de la subunitat beta presentava clars indicis de degradació per part de proteasas i per tant, aquest constructe va ser descartat. Per una altra banda, el constructe amb la HSA fusionada geneticament al C-terminal de la subunitat beta també presentava proteòlisi, però en aquest cas la degradació era parcial i una part de la proteïna es mantenia intacte. Amb la finalitat de trobar les condicions de procés més adequades per tal reduir aquesta degradació parcial de la proteïna es varen realitzar una sèrie de cultius en batch comparant temperatures i pH diferents, determinant a la fi que a pH 6,5 i 22ºC la degradació intracel·lular de la proteïna recombinant es veia clarament reduïda. Ambdós paràmetres es van implementar en un cultiu en fed-batch millorant significativament la quantitat de proteïna no degradada tal com mostren els assajos in vitro. Malauradament, experiments de viabilitat cel·lular duts a terme amb citometria de flux i els anàlisi de les fraccions citosòliques de les cèl·lules deixaven entreveure que la degradació de la proteïna es donava durant la seva via de secreció i que per tant l’eliminació de la proteòlisi mitjançant la implementació d’alternatives en les condicions de procés no era possible. Diferents assajos d’eficàcia de la hormona recombinant es varen dur a terme amb rates immadures demostrant la manca d’activitat LH i FSH de l’hormona recombinant. Sabent que una de les possibles causes per aquesta manca d’activitat podria ser la curta vida mitja de la proteïna en sang, es va dur a terme un estudi farmacocinètic en conills que donava suport a aquest extrem. === During the course of this work, the expression of the heterodimeric hormone (alpha and beta subunits) Pregnant Mare Serum (PMSG), also called equine Chorionic Gonadotropin (eCG), with each subunit under control of AOX1 promoter in the methylotrophic yeast Pichia pastoris, as well as the development of an upstream and downstream process was carried out. First, the development of a cell-based bioassay as well as the cell culture techniques associated with the cell lines was reported. Sertoli cells (TM4) and granulose cells were rejected as a proper cell lines for the bioassay probably because of their poor stereidogenic activity. By contrast, Leydig cells (MLTC-1) resulted in a proper cell line to develop a feasible and reproducible in vitro bioassay to evaluate the hormone bioactivity. Second, two expression vectors with Human Serum Albumin (HSA) fused to C-terminal and N-terminal of PMSG beta subunit, were constructed and subsequently transformed in P. pastoris. Furthermore, both contained a single copy of the PMSG alpha subunit expressed autonomosly. In both constructs, twelve O-glycosylation points located in the C-terminal peptide (CTP) of beta subunit were eliminated in order to reduce the immunogenic effect caused by the mannose-type glycosylation. With the aim to restore a probably reduction of the half-life caused by the CTP deletion, the HSA was genetically fused to beta subunit. Subsequent studies in shake flask revealed a specific cleavage around the linker zone in the construct with the HSA fused to the N-terminal of beta subunit, rejecting this construct as a candidate. On the other hand, construct with HSA fused in the C-terminal revealed also degradation of product, but still intact recombinant PMSG (rPMSG) was recovered and its in vitro activity demonstrated. Thrid, High-cell density cultivation in fed-batch was properly implemented at pH 5.5 and 30ºC. Besides, an adequate two-step purification process was carried out, but a low yield of the entire secreted rPMSG was recovered. Despite this inconvenience, the rPMSG was purified and positively assayed in vitro with MLTC-1 cells. In order to reduce the proteolysis several process approaches were implemented; best working pH to reduce the extracellular degradation of the rPMSG was found at pH 6.5, after 72 hours incubation of purified rPMSG at pH 6,5 almost 66% of the protein remained integral The pH 5.5 and pH 6,5 were compared in batch cultivations at 30ºC, and concluding that intracellularly at pH 6,5 the state of rPMSG improved significantly. Similarly, batch cultivations at 30ºC and 22ºC were performed and clarified that reduced temperature decreased substantially the protease effect intracellularly. Both parameters were applied in fed-batch cultivations with better yields of non-degraded rPMSG as showed in vitro analysis. Unfortunately, cell viability experiments by flow cytometry and cytosolic fraction analysis by western blot pointed out that degradation mainly occurred during the secretion pathway, thus disqualifying any process approach applied to reduce the degradation. Fourth, in vivo assays with immature rats revealed that the purified rPSMG had not FSH and LH activity. Hence, an study of the recombinant hormone half-life was carried out in rabbits and determined that the extremely rapid clearance of protein from the blood-stream could be responsible for deficiencies in the activity.