Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2001-08-20
2002-10-15
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S325000, C435S372000, C424S159100, C210S782000
Reexamination Certificate
active
06465170
ABSTRACT:
The invention relates to a method for depleting viral and molecular pathogens in a biological material containing one or several biological substances to be recovered.
The production of therapeutic proteins and preparations, in particular of immunoglobulin, by extraction from human or animal tissues or liquids, such as blood or plasma, as well as from continuously growing transformed mammalian cells frequently carries the risk of a potential contamination by pathogens, such as viruses, virus-like particles or prions. Therefore, measures must be taken to prevent any pathogens possibly present from being transmitted to human beings.
Human blood or plasma, respectively, may e.g. contain viruses causing diseases such as AIDS, hepatitis B or other hepatitis diseases. With plasma proteins derived from plasma pools the risk of transmitting infectious agents, such as viruses, is very low because of the selection of blood or plasma donations and the production method. Suitable measures, such as excluding high-risk blood donors from donating blood as well as analyzing blood or plasma donations which make it possible to identify infectious donations and exclude them from further distribution, do allow an elimination of most of the infectious donations, yet in most instances not all can be found. Existing assaying systems for detecting infectious viruses in biological materials cannot always completely eliminate concerns regarding a potential transmission of pathogens, since on account of the broad spectrum of infecious pathogens existing it is impossible to assay the starting material for all viruses or molecular pathogens that may be present in a sample. Moreover, most of the tests do not identify the virus itself, but rather identify antibodies developed against that virus so that during the so-called “diagnostic window” a detection of a contamination is not possible. For some groups of viruses, moreover, a reliable or sufficiently sensitive detection method does not exist. Although newly developed assaying methods, in particular nucleic acid amplification methods, such as, e.g., PCR, are highly sensitive and specific, they can only be applied for pathogens whose nucleic acid sequence is known. In those cases in which the human pathogens are known yet a sensitive method of detecting them does not exist, there remains the doubt that a negative result is obtained merely on account of a too low virus content which is below the sensitivity limit of the assaying system.
Therefore, specific removal and/or inactivation methods for depleting viruses have been developed for the production of pharmaceutical and therapeutic products so that infectious particules are no longer to be expected in the final product.
Various inactivation methods are based on a physical-chemical treatment by means of heat and/or chemicals. The methods particularly used are the thermal treatment, pasteurizing, treatment of the protein solution with &bgr;-propiolactone and UV light, treatment with a combination of a solvent and a detergent (so-called S/D method) or exposing the protein solution to light after the addition of a photodynamic substance. With these methods, a virus inactivation of up to 10
6
log steps has been reached. The efficiency of the inactivation method may, however, vary depending on the type of virus present. Although S/D-treated blood products are considered safe in terms of a transmission of HCV, HBV or HIV, non-enveloped viruses, such as HAV or parvovirus, are not inactivated by these methods (Prowse C., Vox Sang. 67 (1994), 191-196).
With biological products, heat treatment methods preferably are carried out either in solution (EP-0 124 506), in the dry state (EP-0 212 040 or WO 82/03871) or in the moistened state (EP-0 324 729). This may often result in losses because of the thermolability of many biological substances.
The type of the inactivation method used may also have an influence on the products, and a stabilisation to minimize the loss of protein thus frequently is required. Moreover, some inactivation methods must be followed by purification steps so as to remove chemicals added.
Methods for virus depletion particularly include chromatographic methods, filtration of protein solutions via a membrane filter, or adsorption of viruses on a solid phase and subsequent removal of the solid phase, as described in EP-0 679 405. However, it has been found that although the treatment with a solid phase, such as, e.g., with Aerosil®, does allow for a removal of HIV up to 4 log steps from an immunoglobulin-containing solution, the loss of IgG may be up to 42% (Gao et al., Vox Sang. 64 (1993), 204-209). With such high losses, such a method appears rather unsuitable for application on a large technical scale.
A widely used chromatographic method for isolating biological substances is anion exchange chromatography. The possibility of depleting viruses by this separation method has also been described in the literature. For instance, virus depletion at an anion exchange chromatography for purifying vWF under conditions under which vWF, yet not the virus, binds to the anion exchanger has been examined (Burnouf-Radosevich, Vox San. 62 (1992), 1-11). By thoroughly washing the column prior to elution, depending on the respective virus, it was possible to recover vWF with a virus depletion of from 1.5 to 5 powers of ten.
Zolton et al. (Vox Sang. 49 (1985), 381-389) examined the virus depletion rate in case of anion exchange chromatography for purifying gamma-globulins under conditions under which gamma-globulins do not bind to an anion exchanger. In these methods, DEAE Sepharose was used as anion exchanger at a pH of 7.5. The infectivity of a starting solution to which hepatitis B virus had been admixed could be eliminated by means of this anion exchange chromatography. By this experiment, a depletion of the hepatitis B virus by the factor 3000 was effected. However, nothing could be said about the depletion rate of other viruses at pH 7.5. What was interesting is that at a pH of below 7.2, viruses appeared in the effluent of the anion exchanger, so that this method generally has been considered not to be applicable in the neutral or weakly acidic pH range.
EP 506 651 describes a multi-step method of recovering a preparation containing IgA, IgG and Transferrin, a reduction of the virus titer having been obtained in each individual method step. During the extraction and precipitation step with 12% ethanol, a virus reduction by a factor of 10
5
could be attained. During the adsorption step, the proteins were bound to an anion exchanger, washed, and eluted again. With this step, virus reduction was at a factor of 10
3
.
Burnouf (Dev. Biol. Stand. 81 (1993), 199-209) reported that during a purification of factor VIII, parainfluenza virus and HIV-1 could be depleted by 4 and 3 powers of ten, respectively, by means of an anion exchange step. When purifying vWF via anion exchange chromatography, a depletion rate of PRV (porcine pseudorabies virus) of 5 log steps has been reported.
Mitra et al. (Curr. Stud. Hematol. Blood Transfus. 56 (1989), 34-43) show that when purifying IgG from plasma according to the plasma fractionating scheme of Cohn-Oncley by a sequence of precipitating steps in the presence of certain concentrations of ethanol and defined pH values in the cold (−5° C.), a virus depletion of >5 and >8 log steps, respectively, of murine C-virus and HIV, respectively, could be obtained. In this paper it is also reported that a 25% ethanol solution at a physiological pH could be highly virucidal. According to Mitra et al., however, a combination of the ethanol treatment with an ion exchange chromatography is neither shown nor suggested.
Hamman et al. (Vox Sang. 67 (1994), 72-77) show that in the course of producing a factor VIII concentrate, a depletion of the virus activity or virus concentration, respectively, of merely 1 to 2 log steps can be attained by means of an ion exchange chromatography.
Therefore, there exists a demand for an industrially applicable method for a guaranteed separation
Barrett Noel
Dorner Friedrich
Eibl Johann
Polsler Gerhard
Baxter Aktiengesellschaft
Heller Ehrman White & McAuliffe
Tate Christopher R.
Winston Randall
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