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Process-scale purification and analytical characterization of highly gamma-carboxylated recombinant human prothrombin.
Wendeler, Michaela; Pabst, Timothy M; Wang, Jihong; Strouse, Robert J; Wang, Xiangyang; Hunter, Alan K.
Afiliación
  • Wendeler M; MedImmune LLC, Department of Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
  • Pabst TM; MedImmune LLC, Department of Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
  • Wang J; MedImmune LLC, Department of Analytical Biotechnology, One MedImmune Way, Gaithersburg, MD 20878, USA.
  • Strouse RJ; MedImmune LLC, Department of Analytical Biotechnology, One MedImmune Way, Gaithersburg, MD 20878, USA.
  • Wang X; MedImmune LLC, Department of Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
  • Hunter AK; MedImmune LLC, Department of Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA. Electronic address: hunterak@medimmune.com.
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.
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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

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