1 Hämosan Life Science Services, Vienna Biocenter, Dr Bohr Gasse 7b, A-1030 Wien, Austria, 2 Leeds General Infirmary, Leeds LS2 9NS, UK and 3 Reinier de graafgroep, loc Diaconessenhuis, Fonteynenburghlaan 5, 2275 CX Voorburg, The Netherlands
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Abstract |
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Key words: gonadotrophin/prion/TSEs/urinary isoforms
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Introduction |
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TSEs are believed to be caused by a self-replicating proteinaceous infectious particle known as a prion (PrPsc). These are pathogenic variants of normal healthy protein isoforms, referred to as cellular prion protein (PrPc). Unlike sporadic or iatrogenic CJD, in nvCJD the infectious agent is distributed in the lymph nodes, spleen and tonsils as well as neural tissue, even in the preclinical phase (Ghani et al., 2000). Speculation has arisen over the presence of abnormal prions in extraneural tissue in nvCJD, which may present an even greater risk of blood infectivity than patients with CJD (Foster, 2000
). It is currently impossible to quantify the risk associated with BSE because of uncertainties regarding the virulence of the BSE strain and the protracted incubation period, which may last up to 3040 years.
The unique characteristic of PrPsc and its unpredictable nature makes the management and control of TSEs all the more difficult. Extra regulatory measures have been recommended concerning blood transfusions since there is a theoretical risk for transmission via plasma-derived products, especially when derived from the UK, which has experienced the highest number of nvCJD cases as a result of the BSE outbreak (Franklin, 1999; Holada et al., 2000
). Although PrPsc infectivity has not been established experimentally, due in part to expectedly low titres in blood and the absence of a homologous human model of infection, it is still imperative that the authorities exercise the utmost caution. There is also new evidence to suggest that a protease-resistant PrPsc isoform exists in the urine of infected animals (Shaked et al., 2001
). Although this finding has yet to be confirmed, it does emphasise the point that all biopharmaceutical products manufactured using substances of animal or human origin carry a theoretical risk of TSE transmission, including pharmaceutical gonadotrophin products. In fact, transmission of PrPsc has occurred during infertility treatment with gonadotrophins derived from human pituitary material, leading to documented mortality from CJD in women between 11 and 14 years after treatment (Cochius et al., 1990
, 1992
; Dumble and Klein, 1992
; Collins and Masters, 1996
). It has been stated that 11 women have died from the disease (B.Lunenfeld, personal communication). Whilst it is well documented that in the past, pituitary-derived preparations were obtained from corpses, it is also important to note that all such products have now been removed from the market.
In order to determine the significance and relevance of PrPsc transmission in these products it is important to conduct risk assessments. These will assess several aspects such as levels of infectivity in the initial source material, the purification steps used to manufacture the product and the daily intake of PrPsc relative to other source materials.
The aim of this paper is to assess the evidence for the transmission of infectious PrPsc in blood and urine, to relate this evidence to fertility treatment with the gonadotrophin preparations menotrophin and recombinant FSH, and to discuss those techniques designed to remove or inactivate PrPsc from biological materials.
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Characteristics of prions |
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Can prions be transmitted by blood and urine? |
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Although there is emerging evidence to suggest that PrPsc-related infectivity can be transmitted from species to species, it is important to interpret the results with caution. Human and animal blood cells display significantly different levels of PrPc, and so species differences must be considered when extrapolating results of rodent transmission to humans (Holada and Vostal, 2000).
A recent study in scrapie-infected mice demonstrated infectivity in the spleen before spreading to the brain via T and B lymphocytes (Weissmann et al., 2001). The lymphocytes themselves did not exhibit infectivity, suggesting that the presence of PrPsc in blood does not necessarily imply that it is infectious. This is supported by evidence that suggests proteinase K sensitive PrPc can be identified on the surface of peripheral blood mononuclear cells in both normal and scrapie-infected sheep (Herrmann et al., 2001
). Although it has not been possible to identify the infectious agent in blood, infectivity can be transmitted via blood. Whole blood taken from a sheep during the symptom-free phase of an experimental BSE infection was shown to transmit infection to another sheep following transfusion (Houston et al., 2000
).
The plasma of mice infected with 301v mouse-adapted BSE was able to transmit infection when injected intracerebrally into healthy mice and provides further evidence that BSE-related agents can be found in the blood or plasma of infected animals (Taylor et al., 2000).
Infectivity bioassays in mice indicated that approximately seven times more plasma and five times more buffy coat are required to transmit infection by the i.v. route compared with the intracerebral route. It was suggested that the lack of epidemiological evidence for the transmission of TSEs from blood in humans is due to the absence of plasma infectivity prior to the onset of clinical symptoms and low levels during the symptomatic stage of the disease. Other factors include the reduction of infectivity by plasma processing steps and the need for higher levels to trigger infection via the i.v. route compared with intracerebral route (Brown et al., 1999).
Exogenous spiking experiments have suggested a minimal risk of acquiring CJD from plasma concentrates since the fractionation steps remove the cellular components of plasma which are associated with the majority of infections (Brown et al., 1998).
Evidence for transmission from urine
The evidence for transmission from urine still needs to be confirmed; however, a recent study demonstrated that a protease-resistant urinary isoform, defined as uPrPsc, can be prepared from the urine of hamsters, humans and cattle affected with TSE (Shaked et al., 2001). However, the implications of this finding are still uncertain because the urinary isoform differs from the brain-derived PrPsc. When hamsters were inoculated with the urinary isoform they did not develop clinical symptoms even after prolonged incubation (270 days), despite the presence of the isoform in their urine. In contrast, hamsters inoculated with brain-derived PrPsc developed clinical symptoms within 80 days and had the urinary isoform of PrPsc in their urine. Some aspects of the methodology may have influenced the appearance of this particular isoform in urine. The study used dialysis to separate and purify the prion; however, this technique may have favoured the production of a soluble transient isoform of PrPsc from normal PrPc, leading to higher than expected levels. It is recommended that the study be repeated without the use of the dialysis technique to establish the presence of infectivity in urine containing uPrPsc. There are also suggestions that certain anionic detergents (i.e. sarkosyl and sodium dodecyl sulphate) are capable of inducing ß-sheet aggregates distinct from PrPsc in terms of infectivity and protease resistance (Xiong et al., 2001
), which may have contributed to the formation of the uPrPsc isoform observed in the Shaked study (Shaked et al., 2001
) which used similar conditions.
Xiong et al. were unable to separate fibrillar and amorphous prion aggregates, the latter of which formed the majority in their analysis (Xiong et al., 2001). This may explain why they were unable to demonstrate proteinase K resistance following detergent/dialysis treatment. By using ultracentrifugation they might have been able to discriminate between the two distinct types of prion. In contrast, Shaked et al. performed ultracentrifugation after dialysis, which enabled them to detect scrapie-associated fibril (SAF)-like prion aggregates (Shaked et al., 2001
). Repeating both studies may determine the influence of ultracentrifugation and dialysis on the formation of proteinase K-resistant prion aggregates.
The transmission of CJD from species to species by urine was demonstrated after urine from a man with CJD was intracerebrally inoculated into mice. However, this result was not repeated when urine from another patient or from a number of infected animals was used (Tateishi, 1985). Furthermore, subsequent investigators have been unable to reproduce these findings and the relatively short incubation period observed in this single case (compared with the time following inoculation with infected brain) suggests that transmission was probably due to cross-contamination within the laboratory (Baron, 1999
). Another early study detected murine CJD infectivity in many tissues including blood, but was unable to confirm any infectivity in urine (Kuroda et al., 1983
). Bioassays of tissue from BSE-infected cattle used to inoculate mice failed to demonstrate infectivity in urine (Scientific Steering Committee, 2002
).
Regarding the transmission of infectivity in urinary-derived gonadotrophins, it is important to consider the fact that proteins are rarely excreted into urine. Normal 24 h protein levels are <150 mg in total (40% albumin, 15% globulins and 40% tissue proteins) and represent the low molecular weight proteins such as microalbumin and -1-acid glycoprotein. The molecular weight threshold for protein excretion is ~40 kDa, which would be expected to exclude the insoluble aggregated PrPsc isoform. However, it seems possible that PrPsc may be present in blood in a non-aggregated form (at concentrations below the recent detection limit), which is subsequently concentrated in the kidneys leading to uPrPsc. The extent of urinary excretion of PrPsc is probably less than that proposed in the Shaked study. PrPsc initially entered the urine as a non-aggregated, protease-resistant isoform, but dialysis appears to have contributed to the formation of a transient semi-aggregated uPrPsc isoform, which concentrates in the urine. The impact of dialysis may have led to higher than expected levels of uPrPsc.
Another source of uPrPsc could originate from the kidney itself. The kidney contains relatively low levels of PrPc (Moudjou et al., 2001), so it is feasible that PrPc derived from the kidney could contribute to the overall uPrPsc levels.
Diagnosing prion infection
Ideally it would be preferable to control the source material and to screen for the presence of PrPsc. However, the very nature of TSEs means that sourcing and screening can only be reliably performed on a retrospective basis. Sourcing urine or blood from so-called safe BSE countries will not guarantee PrPsc-free materials. The BSE status of countries is constantly being updated due to the continuing emergence of new cases in countries previously regarded as being unaffected. There are also limitations to screening since the diagnosis of TSE relies on post-mortem brain samples, although there are a number of diagnostic tools under development which may provide a useful means of identifying infection at earlier stages. One of the problems often encountered is the sensitivity of screening tests. Experimental rodent models suggest a minimum requirement for PrPsc detection in blood buffy coat of <10 pg/ml. The correlation of this level to humans will be dependent on whether infectivity in human blood is lower or greater than rodent experimental models. Some screening tests are within range, so it is feasible that we will be able to detect PrPsc at practical levels in TSE-infected patients blood and possibly urine in the near future (Brown, 2001). It is also important to consider that increasing the sensitivity of a test will be characterized by a rise in the number of false positive results, which may need further validation. Infectivity can be measured by bioassay using either endpoint titration or incubation time assay; however, patterns of infectivity vary according to the method used. These differences can be attributed to variations in the size and number of PrPsc aggregates (Masel and Jansen, 2001
). In-vitro tests are only capable of identifying components that suggest infectivity.
Diagnostic tests used for the surveillance of TSEs currently rely on post-mortem validation by microscopic examination of brain tissue. The current batch of tests available are essentially bioassays of post-mortem tissue, which are time consuming and expensive. Analysis of post-mortem tissue can be performed using a range of techniques, including histopathological examination of the brain, SAF detection by electron microscopy, Western blotting, immunoassay and immunohistochemical detection. It is also important to validate these tests in order to confirm the feasibility, standardization, assay performance, validation criteria, specificity for a particular isoform and predictability of the assay, i.e. it must be able to distinguish between normal and diseased tissue. Although we still rely on post-mortem tests as a means of determining infectivity, there are a number of in-vitro surrogate tests that have been developed which measure levels of proteins known to be associated with TSEs. These surrogate diagnostic tests involve the determination of the 14-3-3 protein, which is located in the cerebrospinal fluid and appears to act as a marker of neuronal cell death (Muller et al., 2000). However, this test is not specific to TSEs as increased levels of this protein are also associated with other diseases such as Alzheimers. Another surrogate test relies on the detection of the ß-protein S-100, in which elevated levels have been demonstrated in patients with genetic and sporadic CJD (Beekes et al., 1999
). However, animal models suggest S-100 is inappropriate for the preclinical detection of scrapie in hamsters, although it may provide a useful in-vitro test for the diagnosis of TSE in naturally or accidentally infected animals.
A number of reliable and sensitive diagnostic tests are now available for BSE-infected animals. Although they are all currently restricted to post-mortem investigation, these tests are being developed and adapted to test for infection in preclinical animals. There are three tests currently in use that have been approved by the European Community: Enfer, CEA-BioRad and Prionics. A detailed summary of each of these tests is outlined in Table I.
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Purification of recombinant versus urinary-derived gonadotrophin |
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Human plasma is already widely used to produce coagulation factors, immunoglobulin preparations and albumins. In the case of gonadotrophins, animal serum features prominently in the production of recombinant gonadotrophin products, and human urine from menopausal women is the source of FSH and LH in the production of human menopausal gonadotrophin (hMG) and highly purified hMG. Therefore, the theoretical risk of infection, albeit negligible, extends throughout the whole spectrum of animal- and human-derived biological materials.
The regulatory authorities have already responded to the potential threat of infection from blood products used for vaccines and have issued guidelines. Currently any bovine-derived material used in the manufacture of a vaccine is regulated according to the Committee for Proprietary Medicinal Products (CPMP) and is continually updated in response to new findings. These measures include exclusion criteria for the source of the plasma, donor deferral criteria and quarantine/withdrawal policies (Committee for Proprietary Medicinal Products, 2001). Such measures are now a prerequisite for products derived from urine as regulatory authorities become aware of the potential for infection. However, the lack of infectivity currently demonstrated in urine is reflected by the CPMP's decision to place urine in the lowest risk category (Committee for Proprietary Medicinal Products, 2001
). In the case of the gonadotrophin preparations, both types of production will have to undergo risk assessment analysis at each stage of production. The biotechnology industry is mainly concerned with validating existing steps in the manufacturing process, which will also apply to the manufacturers of gonadotrophins.
The menotrophins are the established gonadotrophin preparation and have been in use for ~40 years. Their safety record is impeccable and to our knowledge there have been no reports of infectivity with TSEs during this time. The recently introduced highly purified menotrophin (Menopur; Ferring Pharmaceuticals), which does not contain any additional protein other than gonadotrophins, has recently been shown to be comparable with recombinant FSH in terms of safety and tolerability, reflecting the equivalence in purity between both preparations (European and Israeli Study Group on highly purified hMG versus rFSH, 2002). FSH and LH activity are extracted from the urine of post-menopausal women from Argentina, which is known to be a low risk country for BSE and CJD infection. This is a medically well-controlled and fairly static population of donors, which allows for the close monitoring of infectious materials.
However, there are limitations to screening and quarantine as a rapid means of identifying infection, which increases the emphasis on ensuring that there are robust removal or inactivation processes in place.
There at least two potential steps in the recombinant production process that could introduce contamination: FCS, which also contains cell debris and serum proteins, is used as a medium for culturing recombinant cells, and monoclonal antibodies, used in the purification process, which are derived from lymphocytic cells raised in tissue culture medium containing fetal bovine serum.
FCS is derived from a broad population of animals which, theoretically, is likely to be less well protected than humans from the risk of prion infection. The collection of FCS is a rather crude process, which permits the potential for transmission of infectivity from mother to fetus. The use of a bolt to the brain to slaughter the mother disrupts the blood brain barrier allowing the entry of PrPsc into the systemic route. Furthermore, the physical removal of the calf can cause disruption to the placental barrier allowing contamination.
The purification stage using monoclonal antibodies could represent a significant risk for contamination since it is one of the final steps in the production process and the affinity columns cannot be sanitized afterwards to ensure the inactivation of residual infectious materials because the expensive monoclonal antibodies in the column would be destroyed.
In order to validate each step of the manufacturing process for both urinary-derived and recombinant gonadotrophins, the use of a spiking agent which mimics prion proteins is required. The principle of spiking involves a known amount of impurity, which closely resembles the agent of interest, being introduced in a sample to assess the capacity of the purification process to remove or inactivate prions. In the case of PrPsc, it is difficult to obtain a pure sample and this may affect validation.
Several plasma protein purification steps have been investigated in plasma spiked with TSE-infected material. Western blot analyses of PrPsc and bioassay measurements of infectivity revealed that the purification steps were effective at removing PrPsc and associated infectivity (Lee et al., 2001). In support of these findings, 16 plasma fractionation steps spiked with hamster-adapted scrapie showed that the most effective removal techniques included cold ethanol precipitation and depth filtration for albumin and immunoglobulin processes, and ion exchange columns used for the preparation of factors VIII and IX. Hence, the majority of steps are capable of removing abnormal PrPsc (Foster et al., 2000
)
Urine-derived preparations
The purification steps in the production of highly purified hMG involve an initial stage of separation of the gonadotrophin hormones from the urine sample followed by four stages of chromatography to maximize purity (see Figure 1). Validation of each separation technique will be important and may well indicate which are already sufficient to remove PrPsc and associated infectivity.
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Experience obtained in numerous validation studies, although mainly with plasma as a matrix, indicates that ion exchange chromatography (both cationic and anionic exchangers of any type), but not hydrophobic interaction chromatography, tends to remove PrPsc and infectivity. Therefore three of the four chromatographic steps leading to highly purified gonadotrophin preparations from urine have the potential to remove PrPsc. However, all of these steps require thorough validation studies with different types of spiking material.
Given the extremely low possible titre of infectivity in the starting material and the large number of potentially potent removal/inactivation steps, the products derived from urine canpending the results of validation studiesbe considered safe.
Recombinant preparations
The only source of possible contamination with PrPsc is the bovine serum used for freezing and maintaining the recombinant Chinese hamster ovary cell lines and the hybridoma cell lines for producing the monoclonal antibodies used in purification (affinity chromatography), the production media for the recombinant hormones and the recombinant antibodies (see Figure 2). As outlined previously, the risk of contamination with PrPsc is extremely low. Nevertheless, validation studies should be carried out to demonstrate the potential of PrPsc removal in the purification steps. This is of particular importance, as the possible introduction of PrPsc through the monoclonal antibodies used in affinity chromatography purification comes at a late stage of downstream processing.
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One of the more promising techniques that has already demonstrated considerable success is the Haemosafe method used for the inactivation of non-lipid-coated viruses, e.g. polio, and prions (Reichl, 1991). This process can be adapted to treat plasma and serum and is expected to generate similar results with urine. The main steps involve heating the sample in the presence of anionic tensides and stabilizers, which is then followed by incubation with chaotrophic reagents, e.g. urea, which can be conveniently included into existing purification schemes without the need for major investment.
Initial experiments using the Haemosafe technique have already demonstrated the recovery of 83% of hCG in a bioassay (Data on file, 1997). By carefully optimizing the two major steps, even higher recoveries can be achieved. The optimized process will again need validation studies to prove inactivation of PrPsc infectivity.
During the design of validation experiments it is important to not only test the level of inactivation in each step but also to perform an overall validation of the entire process (Reichl et al., 2002). It is also imperative that every manufacturer conducts their own specific validation assessment instead of assuming that a generic production process that has been validated by one manufacturer will apply to another.
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Discussion |
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In relation to the gonadotrophins, the method of production, whether it is extraction from urine or recombinant DNA technology, still utilizes material with a biological origin. PrPsc is now known to cross the species barrier and furthermore is able to infiltrate and propagate in extraneural tissues. While there is growing evidence to suggest that infectivity can be transmitted in blood, the presence of PrPsc in urine and associated infectivity is yet to be confirmed.
In experimental models to date, the unconfirmed infectivity of urine or blood can be explained as either subclinical infection, whereby the animals are merely carriers of the disease, which may or may not be able to transmit infection, or there is a considerably delayed onset of action compared with brain-derived PrPsc.
Risk assessments need to be conducted in both blood- and urine-derived gonadotrophins to determine the actual likelihood of transmitting infection in ovarian stimulation protocols. The relative risk of these products is expected to be very low compared, for example, with the ingestion of meat products. Potential levels of exposure need to be considered and in the case of the gonadotrophin products very small volumes of drug are administered during one treatment cycle, hence the volume and frequency of exposure compared with food intake is negligible. Another factor to consider is the size of the batch used to derive the final product. If infectious PrPsc from a donor is pooled into a much larger volume of urine or plasma then this will be diluted significantly, reducing the chances of exposure. Therefore, when we consider the daily intake of food compared with the comparatively negligible amounts administered during fertility treatment and the purification processes utilized in the production of gonadotrophin preparations, we can conclude that the relative risk of infection is unlikely to be more than theoretical.
There are a number of techniques available to remove or inactivate PrPsc throughout the manufacturing processes, which are applicable to the production of hMG and rFSH. Furthermore, diagnostic tests are under development, which will hopefully be able to rapidly identify preclinical TSEs using blood and urine samples. There are already stringent regulatory measures in place in order to account for viruses and bacteria during the manufacture of gonadotrophins and it is reasonable to assume that such steps are already capable of removing significant amounts, if not all, of PrPsc during the manufacturing process.
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Conclusion |
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Notes |
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References |
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