Expression of recombinant human coagulation factors VII (rFVII) and IX (rFIX) in various cell types, glycosylation analysis, and pharmacokinetic comparison

IntroductionClearance mechanisms for rFVII (or the active enzymerFVIIa) and rFIX are influenced by post-translationalmodifications, especially N-glycosylation. This should beconsidered when choosing a recombinant expressionsystem in view of the varying ability of frequently usedcell lines to perform modifications similar to humanproteins.Differences in the pharmacokinetic properties ofrecombinant FVIIa versus plasma-derived (pd)FVII ordesialylated rFVIIa are known for human FVII(a). AsialorFVIIa clears quickest, whereas pdFVII, having a higherdegree of sialylation, is cleared to a lesser extent [1]. Inthe case of FIX, the degrees ofserine phosphorylationand tyrosine sulfation in the activation peptide havebeen postulated to influence pharmacokinetic behavior,especially in vivo recovery [2].We chose CHO, BHK and HEK293 cells for expres-sion of rFVII to compare post-translational proteinmodifications, and HEK293-derived cell lines to generatehighly phosphorylated and sulfated rFIX for in vivo stu-dies. rFIX from the same clone and production run waspurified using two different down-stream processes: Thefirst to enrich high phosphorylated and sulfated protein,the second to purify total rFIX at high yield. TheseHEK293-derived rFIX isoforms were compared withCHOrFIX and pdFIX in a pharmacokinetic study in FIXknock-out mice.Materials and methods