Pathogen inactivation – regulators aspects
As for plasma derivatives in the early 1980s, in this century pathogen inactivation of single blood components became an option to improve blood safety in a proactive way. And again, any gain in safety has to be counterbalanced by a procedure immanent loss in activity of blood products. The risk of transmitting infectious diseases by transfusion of single blood components is evidently different from treatment with blood products made from large plasma pools. Nonetheless, our experience from the last years shows that transfusion safety becomes increasingly threatened by new emerging infectious agents like West Nile virus in North and Central America, chikungunya virus in Reunion and Italy, dengue fever virus in South America. No testing strategy can avert the safety gap between occurrence and test development for these new pathogens. Thus, being fully aware of the current worldwide epidemiological situation as well as the changed perception of blood safety, regulators should support developments of pathogen inactivation technologies for blood components and escort them until access on the market.
Conditions for marketing authorization
Because of the fact that marketing authorization procedures for single blood components differ considerably within the European Union and also between other countries like USA, Canada, Australia, similar differences apply also to marketing authorization of pathogen-inactivated blood components. In many countries, only a marketing authorization for the medical device used for pathogen inactivation treatment is required; in other countries, blood establishments need a further marketing authorization for the pathogen-inactivated blood component itself. Independently of regulatory procedures, the basic requirements for a pathogen-inactivated blood component prior to its use as a standard transfusion product should be mostly identical. Even if inactivation procedures may be quite different, its evaluation regarding viral safety and toxicology has to follow well-known rules. The evaluation of bacterial inactivation capacity has to follow in principle that for viruses but considering the specific growth behaviour and strain differences of bacteria. It is however nearly impossible to define strict requirements for quality standards as there is no single strong correlation known between laboratory parameters and clinical efficacy of a given component. That means any information on quality and efficacy of the pathogen-inactivated blood component has necessarily to be confirmed by clinical data.
Most of the ICH preclinical safety guidelines have been adopted by the CPMP for the European Union, by the Japanese Ministry of Health, Labour and Welfare (MHLW) and are published in the FDA federal register. It comprises a guideline on the need of carcinotoxicity studies, guidance on testing and dose selection for carcinotoxicity studies, on genotoxicity testing, on pharmacokinetic studies, on toxicity studies including reproductive toxicology, on nonclinical safety studies and on immunotoxicity studies. Even adopted as guidelines for preclinical studies on pharmaceuticals intended for human use, it covers all aspects needed to assess the toxicology properties of pathogen-inactivated blood components.
A prerequisite for any further considerations on pathogen-inactivated blood components is nontoxicity of the inactivation principle as a result from preclinical studies.
Quality data on virus inactivation
Requirements for viral safety evaluation are likewise well described and also laid down in an ICH harmonized tripartite guideline, i.e. adopted by the EU, Japan and the USA. It provides guidance on the choice of ‘model’ viruses, the design and interpretation of studies validating the inactivation and removal of viruses.
Weaknesses of the inactivation procedure which had been discovered during the validation process have to be described in detail. A scarce inactivation capacity against certain viruses may not necessarily lead to refusal of an application, because blood safety is based not only on an effective pathogen inactivation process but also on a thorough donor selection and high quality donor testing. In case of a diminished inactivation capacity, physicians have to be informed appropriately. Thus, for the use of methylene blue/light-treated plasma (MB plasma), information had to be provided on the insufficient effectivity against nonenveloped viruses like HAV and Parvovirus B19.
Quality data on bacteria inactivation
Basically, the same rules can be applied for the validation of bacterial safety evaluation. However, owing to the different growth behaviour of bacteria, great importance must be attached to the exact manufacturing procedure, amongst others storage temperature and time-point of inactivation. The choice of ‘model’ bacteria may be made e.g. according to the Ph. Eur. monograph on sterility testing of cell-based medicinal products. Strains used for validation experiments may comprise ATCC strains which, however, must further be proven to grow in the given blood components and to represent the expected spectrum of bacterial contamination. In contrast to studies with model viruses, spiking experiments with bacteria have to follow two strategies: Spiking with high numbers of bacteria to provide information on inactivation capacity and spiking with very low levels of bacteria to mimic real conditions and to observe the influence of growth characteristics of the individual strains in the manufacturing process. Data interpretation from pathogen inactivation experiments with bacteria should consider the fact that even a minute number of residual not inactivated bacteria may again grow out and cause severe transfusion reactions. Therefore, the inactivation capacity should not only be described in log reduction steps but should also precisely define the manufacturing preconditions for an optimal gain in safety by the inactivation procedure.
In case of certain weaknesses of the inactivation capacity, marketing authorization can be given with appropriate information for physicians. For example, as condition for the marketing authorization of Amotosalen/light-treated platelet concentrates (Amotosalen platelets), the limited capacity against several single species (e.g. Pseudomonas aeruginosa) and the missing capacity against bacterial spores (e.g. Clostridium perfringens, Bacillus cereus) despite its high effectivity against a broad range of gram-positive and gram-negative bacteria had to be pointed out in the physicians information.
Data on blood component quality
The description of manufacturing and product characterization of the inactivating agent has to follow the rules of classical pharmaceuticals for human use.
Testing for evaluation of the product characteristics of blood components should include metabolical, structural and functional properties in comparison with not inactivated components. Results from these experiments are necessary to describe the basic product quality but are not appropriate to draw a clear conclusion on the clinical efficacy of the pathogen-inactivated blood component. In general, the quality of blood components is highly dependent on the manufacturing process and can be influenced e.g. by temperature conditions and time flow during blood withdrawal, centrifugation, separation and storage of the final blood component and of course also by differences in the pathogen inactivation procedure. A meticulous description of the whole blood component preparation is therefore a prerequisite for the assessment of quality data. For example, the observation was made, that three blood establishments using the same medical device and following the instructions of the medical device manufacturer, obtained different product qualities for Amotosalen platelets. Compared to control products, losses in platelet function on day 3 after pathogen inactivation ranged from a nearly unchanged functionality to 20% loss and on day 6 from around 20% up to 65% loss. Looking at the exact manufacturing data, differences were seen in aphaeresis machines, kind of mixing platelets with additive solution and time frame between aphaeresis and inactivation.
The quality not only of cellular components but also that of plasma for transfusion strongly depends on manufacturing details. Large differences are published for the quality of MB plasma mainly because of the use of different systems for treatment. However, even using a well-designed system, which includes a filter for removal of cellular particles and a filter to remove MB photoderivatives after light treatment, and following the instructions of the medical device manufacturer, striking differences in MB plasma quality could be observed between MB plasma manufactured by two blood establishments. Both prepared MB plasma from recovered blood, used nearly identical conditions for centrifugation, separation and inactivation with regard to time–course and temperature. The most evident difference in processing was the time frame between blood withdrawal and centrifugation and between inactivation and deep freezing. The most evident difference in product quality was the higher loss in fibrinogen activity at prolonged (27%) vs. short processing time (14%). It must be noticed that the loss in fibrinogen activity in both cases only occurred following light treatment which means that the quality differences were because of a better maintaining of overall plasma quality at shorter processing times.
Pathogen-inactivated blood components are not produced in large batches and cannot be released by one pharmaceutical plant as well characterized uniform Investigational Medicinal Products (IMP) for its use in a multicenter clinical trial. In the majority of cases, they are manufactured in different blood establishments which may not follow identical procedures. To guaranty an almost homogenous quality for the blood components as IMP, all processing steps have to be exactly defined and precisely described. As delineated, already small deviations in manufacturing may result in different product characteristics and may have an impact on clinical safety and efficacy and may in consequence reduce the validity of a clinical study. Moreover, patients receiving blood components are mostly characterized by multimorbidity. It is therefore of crucial importance to minimize the influence of a variety of different factors on IMP quality, to use a stringent and clear study design, and to realize the clinical trial under GCP conditions.
There exist many examples of inadequately performed Phase III studies. Crucial points for the explanatory power of clinical studies especially for blood components are (1) correct choice of the primary end-point, preferably a clinical and not a laboratory parameter; (2) correct choice of statistical methods (especially in cases with two end-points); (3) clear definition of the objective, preferably noninferiority in the primary end-point; (4) correct choice of control groups; (5) comparability of the untreated and treated component with respect to e.g. platelet count, plasma volume, etc. It is to expect and in most cases also experimentally shown that pathogen inactivation procedures impair the activity of cellular blood components and plasma proteins. It is therefore recommended to use doses of the active substance with an activity comparable to noninactivated components in the treatment arm of Phase III studies.
A stringent application of GCP principles in all study centres is important to guaranty uniform diagnostic evaluation and assessment of adverse reactions, especially required for a valid estimation of safety aspects. Many of the studies carried out so far for pathogen-inactivated components were not optimal designed and insufficiently performed. In consequence, many studies are neither qualified for an assessment of clinical efficacy nor yield robust evidences for product safety and are an immense waste of resources.
Measures following marketing authorization
Blood transfusions are mainly given to severely sick patients, and safety problems may arise which could not be detected in preclinical tests and in clinical studies designed for estimation of efficacy in a well-selected patient group. Moreover, the number of observed patients may be too low to discover very rare adverse reactions like antigenicity formation during long-term treatment. Depending on accumulated knowledge about the safety profile and the kind of inactivation principle, an active postmarketing surveillance should therefore be taken into consideration.
No potential conflicts to declare.