ORIGINAL ARTICLE Treatment Elimination capacity of a TSE-model agent in the manufacturing process of Alphanate Ò /Fanhdi Ò , a human factor VIII/VWF complex concentrate J. M. DIEZ, S. CABALLERO, F. J. BELDA, M. OTEGUI, R. GAJARDO and J. I. JORQUERA Instituto Grifols S.A., R&D Area, Barcelona, Spain Summary. The variant Creutzfeldt–Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE), mainly present in the UK and is associated with the ingestion of bovine products affected with bovine spongiform encephalopathy. Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. We studied the purification steps in the manufacturing process of two FVIII/VWF concentrates (Alphanate Ò and Fanhdi Ò ) in their ability to eliminate an exper- imental TSE-model agent. Hamster scrapie strain 263K brain-derived materials were spiked into sam- ples of the solutions taken before various stages during its production: 3.5% polyethylene glycol (PEG) precipitation, heparin affinity chromato- graphy and saline precipitation/final filtrations. PEG precipitation and affinity chromatography were studied both as isolated and combined steps. TSE agent removal was determined using a labora- tory scale model representative of the industrial manufacturing process. The prion protein (PrP Sc ) was measured with Western blot and TSE infectivity was measured with bioassay. Western blot results were in agreement with those obtained by bioassay, showing a significant removal capacity in the pro- duction process: 3.21–3.43 log 10 for the PEG pre- cipitation; about 3.45 log 10 for the affinity chromatography; and around 2.0 log 10 for the saline precipitation plus final filtrations. PEG precipitation and heparin affinity chromatography were demon- strated to be two complementary TSE-model agent removal mechanisms with total removal being the sum of the two. An overall reduction factor of around 8 log 10 can be deduced. The tests from the production process of FVIII/VWF complex concen- trates have demonstrated their potential for elimi- nating TSE agents. Keywords: Alphanate Ò , factor VIII/VWF complex, Fanhdi Ò , TSE elimination Introduction The variant Creutzfeldt–Jakob disease (vCJD) appears to be associated with the consumption of meat from animals infected with bovine spongiform encephalopathy (BSE). The disease emerged in the UK and was associated with the outbreak among native cattle. Over 183 000 cattle were diagnosed between 1980 and 1996. Depending on the estimations the number of cows affected in UK was 1 million [1] or even much higher, with around 4.5 million cows slaughtered, most of them untested for the presence of the TSE agent [2]. In the rest of the world, from 1980 until the time this study was completed, about 5600 BSE cases were reported in cattle [3]. Most cases of vCJD have occurred in the UK; 168 out of a total of 212 cases worldwide as of April 2009 [4]. The remaining vCJD cases are distributed as follows: one in Canada; 23 in France; four in Ireland; one in Italy; one in Japan; three in Nether- lands; two in Portugal; one in Saudi Arabia; five in Spain and three in the USA. Two of the cases in the USA, two in Ireland and the Canadian and Japanese cases were patients who either resided in or visited UK during the BSE peak. The third USA case was most likely infected in Saudi Arabia [5]. Transmission via blood, plasma or its derivatives has never been observed for the classic forms of Correspondence: J. M. Diez, Instituto Grifols S.A., R&D Area, Barcelona, Spain. Tel.: +34 93 5710 933; +34 93 5710 553; e-mail: josemaria.diez@grifols.com Accepted after revision 20 May 2009 Haemophilia (2009), 15, 1249–1257 DOI: 10.1111/j.1365-2516.2009.02067.x Ó 2009 Blackwell Publishing Ltd 1249