Purification of recombinant DNA-derived factor IX produced in transgenic pig milk and fractionation of active and inactive subpopulations.
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 2-3 g rFIX/(l(-1) h(-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 60 g/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.
Lindsay
Myles
M
Department of Chemical Engineering, 133 Randolph Hall, Virginia Tech, Blacksburg, VA 24061, USA.
Gil
Geun-Cheol
GC
Cadiz
Armando
A
Velander
William H
WH
Zhang
Chenming
C
Van Cott
Kevin E
KE
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004274:DNA, Recombinant / Q000235:genetics; D005164:Factor IX / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine / Q000235:genetics*
D004274:DNA, Recombinant; D008894:Milk Proteins; D011994:Recombinant Proteins; D005164:Factor IX