SOLVENT-DETERGENT TREATMENT OF IgM-ENRICHED IMMUNOGLOBULIN

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1 DARU Volume 11, No. 2, 2003 SOLVENT-DETERGENT TREATMENT OF IgM-ENRICHED IMMUNOGLOBULIN * MOJGAN POURMOKHTAR, ** RASSOUL DINARVAND, * KAMRAN MOUSAVI HOSSEINI, * HOURI REZVAN, * MOHAMMAD ALI JALILI * R&D Department, Iranian Blood Research and Fractionation Co., Tehran, Iran ** Department. of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. ABSTRACT Viral safety of human plasma products plays a key role in their safe uses. Solvent- detergent (SD) virus-inactivation method has gained widespread popularity in the manufacture of biological products. This treatment which inactivates lipid-enveloped viruses effectively consists of incubation of a plasma protein solution in the presence of a non-volatile organic solvent and a detergent. In this study, IgMenriched immunoglobulin was incubated at 24 C for 6 h under slow stirring in the presence of tri(nbutyl) phosphate (0.3% w/w ) as solvent and tween 80 (1% w/w) as detergent. After completion of the inactivation process and removal of the solvent-detergent, the ability of SD-treatment to remove Infectious Bovine Rhinotracheitis (IBR) virus (a lipid-enveloped virus) and Foot-and-Mouth Disease virus (a non-enveloped virus) were evaluated by "virus spiking studies" using a scaled down process. Reduction factor of 4 log was obtained for the SD-treatment of IgM-enriched immunoglobulin spiked with IBR virus. No virus inactivation was observed in the SD-treated IgM-enriched immunoglobulin, spiked with Foot-and-Mouth Disease virus. It was concluded that treatment of IgM-enriched immunoglobulin with TNBP-TWEEN 80 may be considered as an efficient lipid-enveloped virus inactivation step in the manufacture of this product. Key words: Solvent-detergent, Virus inactivation, IgM, Tween 80, Bovine rhinotracheitis virus INTRODUCTION Plasma derivatives which comprise an increasing variety of therapeutic products such as polyvalent immunoglobulins are obtained by pooling plasma of donors. Hence a manufacturing approach that comprises specific measures to ensure very high margin of viral safety is required (1). In other words, viral safety of plasma products plays a key role in their safe uses (2). Fractionation of plasma by ethanol precipitation (3) and protein purification by chromatography (4), at least in part, contribute to the partitioning of viruses and are important tools in optimizing plasma product viral safety(5). However, manufacturers no longer depend on Cohn- Oncley cold ethanol fractionation as the only viral elimination step (6). They have implemented rigorous donor screening plasma testing, good manufacturing practices and additional viral inactivation and validation steps (7) in the manufacturing processes in order to provide viral safety of the plasma products (1,5,8). The most commonly used viral inactivation or elimination treatments are solvent-detergent incubation (9), pasteurization (10), nanofiltration (11), specific dry heat and steam heat treatments, acid ph incubation, combination of beta propiolactone and UV irradiation (8) and photochemical inactivation (1). Since the introduction of modern viral inactivation treatments by solvent-detergent or pasteurization in industry, risks of transmission of the highly pathogenic plasma-borne lipidenveloped viruses (HIV, HBV and HCV) by plasma products have essentially been eliminated (12). The solvent-detergent virus inactivation method has gained widespread popularity in the manufacture of biological products. This popularity arises from several advantages of the treatment such as: its high virucidal action, high protein compatibility, ease of insertion into virtually any pre-existing or newly developed purification process, and the availability of extensive documentation (13). Solvent-detergent treatment consists of incubation of a plasma protein solution in the presence of a non-volatile organic solvent such as tri-(nbutyl) phosphate (TNBP) at a concentration of 0.3% (w/w) and a detergent such as tween 80 at Correspondace: Rassoul Dinarvand, Department. of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran, dinarvan@sina.tums.ac.ir

2 Pourmokhtar et al a concentration of 1% (w/w) (10). This treatment which leads to an efficient viral inactivation by disruption of the structural integrity of lipid-enveloped viruses (14) without alteration in recovery or physiological functions or increase in immunogenicity of plasma proteins (14-16), is generally performed at temperatures between 24 C and 30 C over a maximum of 6 hours under mild stirring (13,17). SD treatment of plasma or immunoglobulin solution followed by virucidal reagents removal by oil extraction and column chromatography, has a proven safety record for Factor VIII products (13) and intravenous immunoglobulins (16). However in most cases SD method is combined with pasteurization at 60 C which ensures inactivation of a wide range of the SDresistant viruses without lipid envelopes (18). Dichtelmuller et al used beta-propiolactone treatme nt for inactivation of viruses in IgMenriched immunoglobulin (8). In this study Solvent-detergent treatment and pasteurization were used for inactivation of enveloped and non-enveloped viruses in IgM-enriched immunoglobulin. The primary target in the fractionation process of each plasma derivatives is inactivation of highly pathogenic plasmaborne lipid-enveloped viruses. An appropriate level of safety against these viruses can be achieved by introduction of techniques involving the use of solvent-detergent treatment and pasteurization (18). MATERIALS AND METHODS IgM-enriched immunoglobulin was produced by cold ethanol fractionation followed by octanoic acid treatment (8). Schematic representation of the manufacturing processes is shown in Fig.1. Reagents: Tri-(n-butyl)phosphate, tween 80, n-hexan, perchloric acid and heptanoic acid were obtained from Merck. Ricinus oil was obtained from Pharm. Raffiniert. Sephadex DEAE A-50 was obtained from Pharmacia Biotech. Viruses: Infectious Bovine Rhinotracheitis (IBR) virus, a member of Herpesviridae family, as a lipidenveloped DNA virus and Foot-and-Mouth Disease Virus (a member of Aphthovirus generation and Picornaviridae family), as a nonenveloped RNA virus, were used for validation of the extent of virus removal during processing. Solvent-detergent treatment The mixture of IgM-enriched immunoglobulin, Human Plasma Cold ethanol fractionation Fraction III Octanoic acid treatment Alcohol precipitation Reconstitution Solvent-detergent treatment Ricinus oil extraction DEAE Sephadex adsorption Virus inactivated IgM-enriched immunoglobulin Fig. 1. Schematic representation of the manufacturing processes for solvent-detergent treated IgMenriched immunoglobulin. tri-(n-butyl)phosphate (0.3% w/w ) and tween 80 (1% w/w) was kept at room temperature (24±2 C) for 6 hours under slow stirring. After completion of the inactivation process, Ricinus oil (5% v/v) was added graduatly under moderate stirring during 45 min. Then the stirring was stopped and the mixture was allowed to stand for 30 min in order to assure a complete separation of the oil and plasma layers and removal of tri-(n-butyl) phosphate. The plasma layer was withdrawn and cleared using 0.45 ìm filter (15). Thereafter, the clear IgMenriched immunoglobulin was passed over a sephadex DEAE A-50 column for the removal of tween 80. Validation of virus inactivation The ability of solvent-detergent treatment to remove relevant viruses was evaluated by virus spiking studies using a scaled down process. IBR virus and Foot-and-Mouth Disease Virus of high titer were added to the IgM-enriched immunoglobulin separately and the manufacturing process was carried out on a laboratory scale. Then ten-fold serial dilutions of samples were made in culture medium and the infectivity titres of cythopatic effects (CPE) inducing viruses were calculated according to Spearman Kaerber (8), which provides the preferred detection system for infectivity determination and virus quantification (8,19).

3 Solvent-detergent treatment of immunoglobulin Virus titre A B Time (min) Fig. 2. Time dependent inactivation of IBR virus by solvent-detergent treatment in IgM-enriched immunoglobulin, A: mixture of IgM-enriched immunoglobulin, TNBP(solvent) and Tween 80 (detergent) spiked with IBR virus, B: IgM-enriched immunoglobulin spiked with IBR virus without solvent-detergent as control RESULTS AND DISCUSSION Table 1 shows results of the process validation studies. In this table, reduction factors (RI) expressed in logarithmic scale which were determined by subtracting the log of the total virus found at the end of the process from which was present initially (9,19), and calculated according to the following equation (20): 10 RI = (V 10 á )/ (V 10 a ) In which, V = Volume of starting material, 10 á = virus titre in starting material, V = Volume of final material, and 10 a = virus titre in final material. The level of virus reduction which is considered acceptable for a single step is 4 log (19). Achievement of a reduction factor of 4 for IBR virus by solvent-detergent treatment of IgMenriched immunoglobulin, proved that the method which was used in this study may be considered as an efficient virus inactivation step in manufactur ing of this product. TNBP-Tween 80 treatment of IgM-enriched immunoglobulin inactivates lipid-enveloped viruses but allows retention of the biologic activity of the proteins highly (15,16). Time-dependent inactivation of IBR virus by tri-(n-butyl) phosphate (0.3% w/w )-tween 80 (1% w/w) treatment is shown in fig.2. The expression of reduction factors (RI) as logarithmic reductions in titre imply that residual virus infectivity may be greatly reduced, but it will never be reduced to zero (20). Table 1. Inactivation of IBR virus in IgM-enriched immunoglobulin by solvent-detergent treatment (TNBP-Tween 80) Treatment Virus Reduction Duration (min) Titre* Factor * These values are means of three tes ts in each step Although ethanol contributes to elimination of viruses both by partitioning and a direct virucidal effect (14), it does not appear to be sufficient to ensure the virus safety of IgMenriched immunoglobulin. However, by incorporation of the additional viral security methods such as solvent-detergent treatment, pasteurization and octanoic acid treatment, this product would appear to be safe and efficacious.

4 Pourmokhtar et al The use of almost any virus, animal or otherwise, can provide useful information in "virus spiking studies". Animal viruses can be selected because of the relevant virus properties (19), e.g. presence or absence of an envelope, size and physico-chemical properties. In this study the process validation studies utilizing spiking experiments with IBR virus and Footand-Mouth Disease virus were performed to give a quantitative estimate virus inactivation s degree by Solvent-detergent treatment of IgMenriched immunoglobulin. IgM-enriched immunoglobulin, spiked with Foot-and-Mouth Disease virus, was treated with tri-(n-butyl)phosphate (0.3% w/w )-tween 80 (1% w/w) and as it was expected, no virus inactivation was observed. Viral validation studies have demonstrated that the processes differ in their capabilities to inactivate lipid and non lipid-enveloped viruses (6). Besides, the International Association of Biological Standardization (IABS) recommends that at least two independent steps for virus inactivation are to be carried out. Therefore, the solvent-detergent method which leads to efficient inactivation of lipid-enveloped viruses need to be combined with pasteurization to ensure inactivation of a wide range of SDresistant viruses without lipid-envelopes (18). REFERENCES 1. Burnouf, T., Radosevich, M. (2000) Reducing the risk of infection from plasma products: Specific preventative strategies. Blood Reviews 14: Josic, D., Schulz, P., Biesert, L., Hoffer, L., Schwinn, H., Kordis-Krapez, M., Strancar, A. (1997) Issues in the development of medical products based on human plasma. J. Chromatogr. B. 649: Roussel, R.H., McCue, J.P. (1991) Antibody purification from plasma. In: Blood seperation and plasma fractionation. Harris, J.R., ed. New york: Wiley-liss, pp: Burnouf, T. (1995) Chromatography in plasma fractionation: benefits and future trends. J. Chromatogr. B. 664: Hellstern, P. (1994) Clinical experience with the viral safety of immunoglobulins. Blood Coagul. Fibrinolysis 5(3): S31-S Ballow, M. (2002) Intravenous immunoglobulins: Clinical experience and viral safety. J. Am. Pharm. Assoc. 42: Committee for proprietary medicinal products (CPMP): AD HOC Working Party on Biotechnology. (1998) Note for guidence on plasma-derived medicinal products. EMEA: Dichtelmuller, h., Rudnick, D., Breuer, B., Ganshirt K.H. (1993) Validation of virus inactivation and removal for the manufacturing procedure of two immunoglobulins and a 5% serum protein solution treated with B-propiolactone. Biologicals 21: Rutler, G.H. (1994) Requirements for safety and quality of intravenous immunoglobulin G preparations. J. Neurol. Neurosurg. Psychiatry 57(suppl): Chandra, S., Cavanaugh, J.E., Lin, C.M.,Piere-Jerome, c., Yerram, N., Weeks, R., Flanigan, E., and Feldman, F. (1999) Virus reduction in the preparation of intravenous immune globuline: in vitro experiments. Transfusion 39: Burnouf-Radosevich, M., Appourchaux, P., Huart, J.J., Burnouf, T. (1994) Nanofiltration, a new specific virus elimination method applied to high-purity Factor IX and Factor XI cocentrares. Vox. Sang. 67: Tabor, E. (1999) The epidemiology of virus transmission by plasma derivatives: Clinical studies verifying the lack of transmission of hepatitis B and C viruses and HIV type 1. Transfusion 39: Horowitz, B., Prince, A.M., Hamman, J., Watklevicz, C. (1994) Viral safety of solvent / detergent-treated blood products. Blood Coag. Fibrinolysis 5(3): S21- S Eriksson, B., Westman, L., Jernberg, M. (1994) Virus validation of plasma-derived products produced by Pharmacia, with particular referance to immunoglobulins. Blood Coag. Fibrinolysis 5(3): S37-S Hellstern, P., Sachse, H., Schwinn, H., Oberfrank, K. (1992) Manufacture and in vitro characterization of a solvent/detergent-treated human plasma. Vox. Sang. 63:

5 Solvent-detergent treatment of immunoglobulin 16. Chidwick, K., Matejtschuk, P., Gascoigne, E., Briggs, N., More, J., Dash, C. (1999) Clinical experience with a new solvent detergent-treated intravenous immunoglobulin free of hypotensive effects. Vox. Sang. 77: Mould, G.P., Sutton, J.A., Matejtschuk, P., Gascoigne, E.W., Dash, C.H. (2001) Solvent / detergent treatment does not alter the tolerance or uptake of human Normal Immunoglobulin for injection. Vox. Sang. 80: Viral elimination or inactivation methods. (1999) In: Plasma fractio nation programmes for developing countries: Technical aspects and infrastructural requirements. WHO Regional Publications, Eastern Mediterranean series. pp: Roberts, P. (1996) Virus safety of plasma products. Medical Virology 6: Committee for proprietary medicinal products: AD HOC working party on biotechnology/pharmacy and working party on safety of medicines. (1991) Note for guidance on validation of virus removal and inactivation procedures. Biologicals 19: