Meinhard Hasslacher

Mutation of the surface‐exposed amino acid Trp2313 to Ala in the FVIII C2 domain results in defective secretion of the otherwise functional protein

The C2 domain of factor VIII (FVIII) is important for FVIII–phospholipid (PL) and FVIII–von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain–PL interface. Within loop four, the solvent‐exposed amino acid, Trp2313, is believed to contribute to FVIII–PL binding. To analyse this interaction, the amino‐acid exchange Trp2313 to Ala (W2313A) was introduced into the C2 domain of B‐domain‐deleted FVIII (dBFVIII). Both proteins, dBFVIII and W2313A, were expressed in a mammalian expression system. Labelling experiments showed that the mutation W2313A resulted in reduced secretion but did not affect intracellular synthesis of the protein. Specific activity, kinetic parameters, binding to VWF and haemostatic potential in a murine model of haemophilia A were found to be similar for both proteins. Binding studies to synthetic 4% phosphatidyl‐l‐serine vesicles showed, however, a 28‐fold higher KD for W2313A, indicating the important role of Trp2313 in the FVIII–PL interaction. In conclusion, the C2‐domain‐surface‐exposed residue Trp2313, is critical for secretion of the protein. The W2313A mutation weakens binding to phosphatidyl‐l‐serine vesicles but the mutant protein has the same effector function as dBFVIII in vitro and in vivo.

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