Ion exchange q sepharose 4ff

Example 9

Cloning of pSYN-FVII-064

Synthesis of DNA sequence from HindIII to EcoRI of pSYN-FVII-064 was outsourced and cloned into HindIII/EcoRI sites of pBUDCE4.1 (Invitrogen)

Expression and Purification of FVII-064

DNA was transiently transfected as described herein. The transfection DNA contained PC5 to fully process the cscFc linker, described in the protein sequence, connecting the C-terminus of the first Fc moiety to the N-terminus of the second Fc moiety

Characterization of Cleavage of cscFc Linker of FVII-064

FVII-064 was purified using Ion Exchange: Q sepharose 4FF from GE healthcare. Secondary capture was performed using shFcRn (soluble human FcRn) affinity (NHS-coupled shFcRn with sepharose 4FF beads). All steps were performed at 150 cm/hrs linear Flow Rate. FIG. 5 illustrates SDS PAGE analysis of FVII-064 following transient expression (cotransfected with PC5) and purification. Under reducing conditions we observe 2 distinct bands for FVII-Fc and Fc, demonstrating full cleavage of the cscFc linker

Example 9

Cloning of pSYN-FVII-064

Synthesis of DNA sequence from HindIII to EcoRI of pSYN-FVII-064 was outsourced and cloned into HindIII/EcoRI sites of pBUDCE4.1 (Invitrogen)

Expression and Purification of FVII-064

DNA was transiently transfected as described herein. The transfection DNA contained PC5 to fully process the cscFc linker, described in the protein sequence, connecting the C-terminus of the first Fc moiety to the N-terminus of the second Fc moiety

Characterization of Cleavage of cscFc Linker of FVII-064

FVII-064 was purified using Ion Exchange: Q sepharose 4FF from GE healthcare. Secondary capture was performed using shFcRn (soluble human FcRn) affinity (NHS-coupled shFcRn with sepharose 4FF beads). All steps were performed at 150 cm/hrs linear Flow Rate. FIG. 5 illustrates SDS PAGE analysis of FVII-064 following transient expression (cotransfected with PC5) and purification. Under reducing conditions we observe 2 distinct bands for FVII-Fc and Fc, demonstrating full cleavage of the cscFc linker