Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII

Abstract

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of f1 AM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3 -4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials. B 2004 Wiley Periodicals, Inc.

Clinical evaluation of moroctocog alfa (AF‐CC), a new generation of B‐domain deleted recombinant factor VIII (BDDrFVIII) for treatment of haemophilia A: demonstration of safety, efficacy, and pharmacokinetic equivalence to full‐length recombinant factor VIII

“…Moroctocog alfa (AF‐CC), herein referred to as BDDrFVIII (B‐domain deleted rFVIII), is manufactured by an albumin‐free cell culture process using Chinese hamster ovary (CHO) cells grown in a chemically defined serum‐free cell culture medium that contains no materials derived from human or animal sources [2]. A synthetic peptide affinity ligand (TN8.2) replaces the murine monoclonal antibody used in ReFacto affinity chromatography, and an additional 35 nm viral filtration step is added during purification, expanding upon the viral‐safety programme currently in use for ReFacto [3].…”

West Nile Virus inactivation by the solvent/detergent steps of the second and third generation manufacturing processes for B‐domain deleted recombinant factor VIII

“…[13] rather than a hybridoma cell line-derived antibody as is used in BDDrFVIII manufacture, thus providing further viral safety assurance [14][15][16]. First, HSA is not employed as a medium supplement and no other human-derived proteins are used in BDDrFVIII AF manufacture.…”

An improved manufacturing process for Xyntha/ReFacto AF

“…The peptide has an acetylated N‐terminus and no primary amines, with the exception of a C‐terminal lysine residue. This allows for efficient, site‐directed immobilization of the peptide via the C‐terminal lysine by the amine reactive N‐Hydroxysucccinimide immobilization chemistry [4].…”

“…Figure 4 shows the binding isotherm for the immobilized peptide and BDDrVIII. The data show excellent fit to a Langmuir isotherm ( r 2 = 0.99), indicating a single mode of interaction, without protein‐protein interactions, and a dissociation constant of 0.92 μ m [4].…”