title: Assessment of the impact of manufacturing changes on the physicochemical properties and biological activity of Her1-ECD vaccine during product development.
abstract: Vaccine preparations based on the extracellular domain of Her1 (Her1-ECD) have demonstrated, in vitro and in vivo, a potent antimetastatic effect on EGFR(+) Lewis lung carcinoma model, while associated side effects were absent. The Her1-ECD is a glycoprotein with a molecular weight of 105 kDa and has 11 potential sites for N-glycosylation. Currently Her1-ECD based vaccine has been evaluated in patients with hormone refractory prostate cancer. Her1-ECD molecule used for in clinical trials was obtained from culture supernatant of HEK 293 transfectomes used the protein free culture media and is purified by immunoaffinity chromatography. In order to increase the cell growth and productivity, new defined culture media have been developed (alternative culture media) in Her1-ECD vaccine production process. In this work, a comparability study was performed to evaluate the impact of process changes in the characteristics physic-chemical and biologicals of the Her1-ECD protein and the degree of similitude between both variants. Techniques such as: SDS-PAGE, SEC-HPLC, isoelectric point, peptide mapping, mass spectrometric, SCX-HPLC, oligosaccharide map, ELISA and flow cytometric were used with this aim. Results indicated that this process change decreases the degree of sialylation of the protein but does not affect its biological activity (measured as titers of Abs and recognition for A431 cell line).
authors.affiliation: Development Process Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: katiag@cim.sld.cu.
authors.forename: Katia
authors.initials: K
authors.lastname: Garcia Duardo
authors.affiliation: Development Process Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: yadirap@cim.sld.cu.
authors.forename: Yadira
authors.initials: Y
authors.lastname: Prieto Curbelo
authors.affiliation: System Biology Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: judithr@cim.sld.cu.
authors.forename: Judith
authors.initials: J
authors.lastname: Raymond Pous
authors.affiliation: Development Process Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: yamiletr@cim.sld.cu.
authors.forename: Estela Yamilet
authors.initials: EY
authors.lastname: Rabasa Legón
authors.affiliation: Tumoral Immunology Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: belinda@cim.sld.cu.
authors.forename: Belinda Sánchez
authors.initials: BS
authors.lastname: Ramírez
authors.affiliation: Development Process Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: katiar@cim.sld.cu.
authors.forename: Kathya Rashida de la Luz
authors.initials: KR
authors.lastname: Hernández
authors.affiliation: Development Process Direction, Center of Molecular Immunology, 216 Street and 15th Avenue, Atabey-Siboney, Playa, P.O. Box 16040, Havana 11600, Cuba. Electronic address: adolfo@cim.sld.cu.
authors.forename: Adolfo
authors.initials: A
authors.lastname: Castillo Vitoch
mesh_terms: D000818:Animals; D019496:Cancer Vaccines / Q000276:immunology / Q000592:standards*; D018827:Carcinoma, Lewis Lung; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000737:chemistry* / Q000276:immunology*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000276:immunology*; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D019496:Cancer Vaccines; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Comparability studies; EGFR; HPLC; Mass spectrometry; Recombinant protein