Purification of recombinant DNA-derived factor IX produced in transgenic pig milk and fractionation of active and inactive subpopulations

Myles Lindsay, Geun Cheol Gil, Armando Cadiz, William H. Velander, Chenming Zhang, Kevin E. Van Cott

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

Transgenic animal bioreactors can be engineered to make gram per liter quantities of complex recombinant glycoproteins in milk. However, little is known about the limitations in post-translational processing that occurs for very complex proteins and how this impacts the task of purification. We report on the purification of recombinant factor IX (rFIX) from the milk of transgenic pigs having an expression level of 2-3g rFIX/(l-1h-1), an expression level that is about 20-fold higher than previously reported. This purification process efficiently recovers highly active rFIX and shows that even complex mixtures like pig milk, which contains 60g/l total endogenous milk protein and multiple subpopulations of rFIX, can be processed using conventional, non-immunoaffinity chromatographic methods. Without prior removal of caseins, heparin-affinity chromatography was used to first purify the total population of rFIX at greater than 90% yield. After the total population was isolated, the biologically active and inactive subpopulations were fractionated by high-resolution anion exchange chromatography using an ammonium acetate elution. Capillary isoelectric focusing of the active and inactive rFIX fractions demonstrated that the active subpopulations are the most acidic.

Original languageEnglish (US)
Pages (from-to)149-157
Number of pages9
JournalJournal of Chromatography A
Volume1026
Issue number1-2
DOIs
StatePublished - Feb 13 2004

Keywords

  • Factor IX
  • Heparin
  • Milk
  • Proteins
  • Tansgenic animals

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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