Elsevier

Biologicals

Volume 31, Issue 3, September 2003, Pages 165-173
Biologicals

Manufacturing process of Anti-thrombin III concentrate: viral safety validation studies and effect of column re-use on viral clearance

https://doi.org/10.1016/S1045-1056(03)00035-6Get rights and content

Abstract

A manufacturing process for the production of Anti-thrombin IIII concentrate is described, which is based primarily on Heparin Sepharose affinity chromatography. The process includes two sequential viral inactivation/removal procedures, applied to the fraction eluted from the column, the first by heating in aqueous solution at 60 °C for 10 h and the second by nanofiltration. Using viral validation on a scaled-down process both treatments proved to be effective steps; able to inactivate or remove more than 4 logs of virus, and their combined effect (>8 logs) assured the safety of the final product. Viral validation studies of the Heparin Sepharose chromatographic step demonstrated a consistency of the affinity of the resin for viruses over repeated use (16 runs), thus providing evidence of absence of cross-contamination from one batch to the next. It was concluded that the process of ATIII manufacturing provides a high level of confidence that the product will not transmit viruses.

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:

  • 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:

  • 1.

    Virus inactivation study of pasteurisation step.

  • 2.

    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

  • 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.

  • 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.

  • 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)

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