Manufacturing process of Anti-thrombin III concentrate: viral safety validation studies and effect of column re-use on viral clearance
Introduction
Anti-thrombin III (ATIII) is a single-chain glycoprotein with an approximate molecular weight of 58 kDa which is synthesised in the liver and is present in plasma at a concentration of about 150 mg/l. Its physiological function is to act as the main inhibitor of blood coagulation and it is especially recognised as the principal plasmatic antagonist of thrombin. Reduced levels of ATIII, lower than 80% than normal, may predispose a hyper-coagulable state, which may progress into localised or disseminated thrombotic disorders [1], [2]. The availability of ATIII concentrates permits an efficient treatment for congenital or acquired ATIII deficiencies [3]. Purified preparations of ATIII have been obtained as a result of the use of chromatographic procedures together with the manufacturing process. Our manufacturing process uses a combination of two chromatographic techniques, ion exchange DEAE–Sephadex and Heparin Sepharose affinity chromatography, to produce a purified ATIII solution. Furthermore, a great deal of progress has been made during recent years on the safety aspect, both by improving the safety of the plasma pool (donor screening, donor look-back, post-collectiontesting) and by the development of specific virus inactivation/removal protocols. Incorporating effective validated steps for viral inactivation/removal in the manufacturing process of plasma derived medicinal products has been highly recommended [4], [5]. Our manufacturing process for the purification of ATIII includes two steps aimed to increase viral safety, the first is by viral inactivation through pasteurisation at 60 °C for 10 h and the second is by viral removal through nanofiltration.
The purpose of this study was to determine the ability of the pasteurisation step to inactivate and of the nanofiltration step to remove a wide range of viruses of different physico-chemical characteristics. Moreover, process validation studies were conducted on the scaled-down manufacturing process to establish the effect of the repeated use of the affinity chromatography resin which cannot be sterilised by heat treatment. Failure of the NaOH sanitisation treatment to remove or decontaminate viruses between subsequent runs can result in the accumulation of viruses in the resin column. Virus particles could be eluted during subsequent runs, leading to a reduction in overall viral safety of the product.
Section snippets
ATIII purification process
The ATIII manufacturing process includes purification from pro-thrombin complex supernatant by affinity chromatography (Heparin Sepharose 6FF, Pharmacia), pasteurisation for 10 h at 60 °C of the ATIII solution eluted from the column, diafiltration/concentration, nanofiltration, sterile filtration, filling and freeze-drying.
The nanofiltration step consists of two sequential filtrations through virus removing nanofilters at 35 nm nominal pore-size pre-filter, followed by a 15 nm nominal pore-size
Virus strains and assay systems
The following virus strains–host cell system were used:
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Inactivation step:
human immune deficiency virus (HIV-1) (Strain RF) and C8166 (human T-lymphoblastoid) cells, obtained from the Medical Research Council Collaborative Centre, London, UK;
bovine viral diarrhoea virus (BVDV) (Strain NADL) and BT cells, obtained from the ATCC, Rockville, Maryland, USA;
pseudorabies virus (Psrv) (Strain Aujeszky) and Vero (African green monkey kidney) cells, obtained from the Collection of Animal Cell Cultures,
Viral safety validation studies
Three viral safety validation studies have been carried-out:
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Virus inactivation study of pasteurisation step.
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Virus removal study of the Planova 35™/Planova15™ membrane filtration procedure.
- 3.
Viral validation study for the re-use of Heparin-Sepharose column.
The experimental design of the validation studies was performed according to the European Guidelines issued by the Committee for Proprietary Medicinal Products (CPMP) [10]and following Good Laboratory Practices duly certified by regulatory
Down-scaling validation
In each study, three downscaled experiments of the step under evaluation were performed and data of the relevant parameters were compared to the corresponding data of the industrial process. The column volume in manufacturing scale was of 50,265 ml and the downscaled version for validation was on a volume of 17.7 ml (downscaling factor was 2839). The elution profile of down-scaled process for the spiking experiments (Fig. 2) is comparable to that obtained without virus spike and in the
Product characterisation
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The fraction of ATIII able to bind to heparin is not less than 60%, as determined by two-dimensional immunoelectrophoresis and as established by the European Pharmacopoeia 1997 [13]for therapeutic concentrates of ATIII.
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The SDS–PAGE (Fig. 3) showed, under reducing conditions, the presence of a single band with an approximate molecular weight of 66 kDa. Under non-reducing conditions the band presented a slight change of mobility resulting in an approximate molecular weight of 58 kDa.
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The
Discussion
In the Anti-thrombin III manufacturing process there are two specific viral inactivation/removal steps designed to target enveloped viruses and small non-enveloped viruses respectively.
The step of pasteurisation was validated for the inactivation capability of four viruses: HIV-1, BVDV, PsRV, Reo type 3. All tested viruses were quickly inactivated and the reduction for each virus was >4 logs. This step is therefore considered to be an effective viral inactivation step.
The results obtained from
Acknowledgements
The authors thank Prof. G. Vicari for review of the manuscript and most valuable remarks.
References (16)
- et al.
Some properties of antithrombin III and its concentration in human plasma
Thromb Res
(1980) - et al.
Analysis of anti-thrombin III microheterogeneity by isoelectric focusing in polyacrylamide gels and immuno blotting
Throm Res
(1985) - et al.
Antithrombin and related inhibitors of coagulation proteinases
- et al.
Antithrombin III deficiency and thromboembolism
Clin Hematol
(1981) - The European Agency for the Evaluation of Medicinal Products: Human Medicines Evaluation Unit. Committee for...
- Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma...
- et al.
Investigations on anti-thrombin III in normal plasma and serum
Br J Haemat
(1975) - Development technique file nos 100, 130 and 200, PhastSystem User Manual Amersham Pharmacia Biotech, Uppsala, Sweden,...
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