Large‐Scale Gel Filtration Chromatography for the Production of a Solvent/Detergent‐Treated High‐Purity Factor VIII Concentrate

Abstract

To produce a tri(n-butyl)phosphate/sodium-cholate-treated intermediate-purity factor VIII (FVIII) concentrate with a specific activity of about 1 IU/mg, we used a simple gel filtration step with Sephadex G25 to remove the solvent/detergent reagents from the final product. By exchanging the Sephadex G25 gel for a new high-resolution gel (Sephacryl S400 HR), we obtained a high-purity FVIII concentrate, by simultaneous elimination of about 98% of the extraneous proteins and removal of the solvent/detergent reagents, without reducing the FVIII:c yield and without altering the production scheme. With different protein analysis techniques we analysed the resulting FVIII concentrate and, comparing it to the formerly produced intermediate-purity FVIII concentrate, demonstrated improved purity.

Preparation of Intermediate- Purity Factor VIII Concentrate by Direct Gel Filtration of Cryoprecipitate

“…In Dengler et al [7], the large increase in puri fication factor is primarily due to the use of S-400HR in the last desalting step. In both this method and Dengler et al's [7], the value given for the enrichment factor does not include the added albumin. Although a direct comparison …”

“…Each method gave a different recovery of FVIII in their cryoprecipitate from plasma, hence the table only compares the increased purification relative to cryoprecipitate. Benny et al [9] used the CPG method of Margolis and Rhoades [2] whereas the rest [7,15,16] were selective precipitation methods. In Dengler et al [7], the large increase in puri fication factor is primarily due to the use of S-400HR in the last desalting step.…”

“…Benny et al [9] used the CPG method of Margolis and Rhoades [2] whereas the rest [7,15,16] were selective precipitation methods. In Dengler et al [7], the large increase in puri fication factor is primarily due to the use of S-400HR in the last desalting step. In both this method and Dengler et al's [7], the value given for the enrichment factor does not include the added albumin.…”

“…These gel filtration media are not suit able for large-scale gel filtration because of their low flow properties and low structural strength. A recent report [7] described the use of a gel filtration media, Sephacryl S-400HR (Pharmacia), instead of Sephadex G25 (Phar macia) in the final large-scale desalting step as an adapta tion of a method reported by Horowitz et al [8]. We de cided to investigate the feasibility of using Sephacryl S-400HR (fractionation range of 20-8,000 kD), as well as another new gel filtration media, Sephacryl S-500HR (Pharmacia; fractionation range of 40-20,000 kD, in the purification of FVIII.…”

“…In most viral inactivation steps using solvent/detergent treatments, sodium cholate was the preferred detergent due to the ease of removal in the final desalting step using Sephadex G25 [7,20]. In this case, 1% TNBP with 1% Triton X-100 is the preferred solvent/detergent combina tion for the viral inactivation of cryoprecipitate because of their greater efficiency at viral inactivation as well as a greater recovery of FVIII activity [20,21].…”