Journal of Chromatography A, 1026 (2004) 149–157 Purification of recombinant DNA-derived factor IX produced in transgenic pig milk and fractionation of active and inactive subpopulations Myles Lindsay a , Geun-Cheol Gil a , Armando Cadiz b , William H. Velander a,1 , Chenming Zhang c , Kevin E. Van Cott a,∗ a Department of Chemical Engineering, 133 Randolph Hall, Virginia Tech, Blacksburg, VA 24061, USA b Finlay Institute, Havana, Cuba c Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA Received 25 July 2003; received in revised form 4 November 2003; accepted 5 November 2003 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 2–3 g rFIX/(l -1 h -1 ), an expression level that is about 20-fold higher than previously reported. This purification process efficiently recovers highly ac- tive 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 bio- logically 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. © 2003 Elsevier B.V. All rights reserved. Keywords: Milk; Tansgenic animals; Factor IX; Heparin; Proteins 1. Introduction Hemophilia B is a bleeding disorder caused by a con- genital deficiency in circulating factor IX activity. The ideal treatment of hemophilia B is prophylactic treatment, which significantly reduces complications and improves the qual- ity of life [1]. Unfortunately, this regimen is unavailable for many hemophiliacs because the only sources of replace- ment factor IX are plasma-derived factor IX (pd-FIX) and recombinant factor IX (rFIX) produced in Chinese hamster ovary (CHO) cells (BeneFIX) [2]. Pathogen safety concerns with pd-FIX, combined with the limited supply of pd-FIX and rFIX from CHO cells make prophylactic treatment of hemophilia B expensive. Safer and more productive sources of rFIX are thus desired. ∗ Corresponding author. Fax: +1-540-231-5022. E-mail address: kvancott@vt.edu (K.E. Van Cott). 1 Department of Chemical Engineering, University of Nebraska, Lincoln, NE 68588, USA. Factor IX is a complex protein with numerous post- translational modifications (PTMs). These PTMs are required for biological activity and pharmacokinetics, and include propeptide removal, vitamin K-dependent -carboxylation of Glu to -carboxyglutamate (Gla), Ser- phosphorylation, Tyr-sulfation, and glycosylation [3]. In par- ticular, -carboxylation and propeptide removal are required for the procoagulant activity of factor IX (Fig. 1). The com- plexity of these multiple PTMs also necessitates production of rFIX in mammalian cells. However, not all mammalian cells are equally proficient in performing all of these PTMs. For example, CHO cells were shown to have significant rate limitations in propeptide removal, -carboxylation, and Ser-phosphorylation [4–6]. Additionally, transgenic sheep were not able to produce any significant amounts of biolog- ically active rFIX in milk [7]. We have previously published immunoaffinity methods for purification of biologically ac- tive rFIX from transgenic pig milk having lower expression levels of 100–200 g/ml containing 35–70 U/ml [8]. The rFIX was fully -carboxylated at this level of production. 0021-9673/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2003.11.006