Process-scale purification and analytical characterization of highly gamma-carboxylated recombinant human prothrombin.
J Chromatogr A
; 1325: 171-8, 2014 Jan 17.
Article
en En
| MEDLINE
| ID: mdl-24369997
ABSTRACT
Prothrombin (coagulation Factor II) is a complex multidomain glycoprotein that plays a central role in blood coagulation. It is the zymogen precursor to the protease thrombin that catalyzes the formation of the fibrin clot and regulates a multitude of other cellular responses related to coagulation and hemostasis. For the biological activity of prothrombin, the vitamin K dependent posttranslational modification of glutamic acid residues to gamma-carboxylglutamic acid is of crucial importance. Prothrombin can be recombinantly expressed using mammalian cell culture. However, the product is a heterogeneous mixture of variants with different degrees of carboxylation, requiring separation of closely related charge isoforms. A second challenge for purification is the need to remove traces of the product-related impurity thrombin, a protease, to extremely low levels. In this work, we describe a purification strategy that provides solutions to both challenges and results in an efficient and robust process for active recombinant prothrombin. We also describe the analytical characterization of recombinant prothrombin by HPLC, LC-MS/MS, and complementary biochemical assays.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Protrombina
/
Procesamiento Proteico-Postraduccional
/
Cromatografía Líquida de Alta Presión
/
Espectrometría de Masas en Tándem
Límite:
Humans
Idioma:
En
Revista:
J Chromatogr A
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos