Virus control
What about killing the virus and bacteria?
By Dicam Microwaves APS microwave plants it is guaranteed that the trays and pallets will be heated homogeneously up to minimum 90°C. It has been tested by SIK (The Swedish Institute for Food and Biotechnology), that the killing effect is 10-6 CFU Serratia Marcescens. (Serratia Marcescens has been used as an indicator organism to avoid work with actual pathogens as e.g. Salmonella. Serratia Marcescens is, as Salmonella, a member of the family Enterobacteriaceae).
According to the Danish Veterinary Institute (Danish Ministry of Food, Agriculture and Fisheries) Serratia Marcescens is not usable for verifying the effect on Avian Influenza Virus (AIV) and Newcastle Disease Virus (NDV), but earlier studies show that neither of these viruses are considered heat stable, and consequently heat inactivation of them is feasible under practical conditions.
The heat stability of NDV and AIV is considered similar, and it is earlier proven by Foster et al. (1957) that the NDV can be inactivated at 70°C in less than 60 sec. According to OlE AIV is inactivated in 3 hours at 56°C but only 30 minutes already at 60°C. (text from the SIK report UP 98-9819)
High possibility
for re-use of the paper trays by using the DICAM MICROWAVESAPS microwave technology
Besides the big advantage by inactivating virus and killing bacteria it has
in practice been seen that the trays can be reused more times than earlier.
Reason is the removal of water and moisture content in the paper tray, which
after each process dry up the tray so it becomes strength as good as a new one.
Combination between a good quality of egg tray and heat treatment has shown
in practice that it is possible to reuse the trays until normal wear and tare
destroys the trays.
Ultra Short
Time Heating with Microwaves
Advancing the accepted concept of high temperature - short time heating for
viral inactivation, Dicam Microwaves has devised systems where heat exposure
at peak temperature is in milliseconds rather than seconds. This ultra short
time heating permits the preferential denaturing of viruses while maintaining
the molecular structure and biological activity in heat sensitive material.
Separation
of clean and dirty trays
To secure an effective hygienic it is important to focus on the total logistic,
only installing a microwave system to inactivate viruses and kill bacteria does
not provide a secure process. The individual customer is recommended to make
an effective sorting of the trays before pasteurization to discard dirty trays.
This can be done automatic by using the TrayChecker. It is also important to
divide the storage of trays and pallets into a dirty - and a clean zone.
Monitoring
the pasteurisation process
In the Dicam Microwaves APS software, the accumulated number of working hours,
etch batch of treated trays and pallets are monitored, and can be printed out
from the PC there are connected to the system. It is there for easy for the
customer and the local Veterinarian Service to control if there is a connection
between the pasteurized items and the total use of items by the individual users.
The results:
Virus Inactivation
Virus chlamydia
The goal of virus inactivation is the irreversible loss of viral infectivity.
Inactivation of virus is superior to removal: the exact and careful determination of the inactivation kinetics allows for a highly reliable and precise performance and validation of the inactivation process.
In general, non-invasive physical methodologies such as High Temperature Short Time (•HTST•) heat treatment are preferred over invasive methodologies using chemical agents, which have to be removed and require expensive analytical monotoring.
The loss of the infectivity does not necessarily mean the complete destruction or disintegration of the virion - which might be most desirable -, but is already given by the irreversible denaturation of distinctive viral components which are essentially required by the virion in order to be able to infect a host cell. Hence, the inactivation procedure should again cover a broad range of virus species featuring various degrees of resistance. Some popular inactivation processes like the solvent-detergent (S/D-) treatment or acid treatment are solely feasible for a limited range of virus species; small non-enveloped viruses such as Polio, SV40 and Parvo (B19) remain unaffected.
HTST Virus Inactivation
The introduction of High Temperature Short Time (•HTST•) heat treatment
offers for the first time a substantial inactivation of small non-enveloped
viruses while fully maintaining the integrity of the product. With respect to
the concept of validation of viral clearance, the High Temperature Short Time
(•HTST•) heat treatment features the unique opportunity to spike
and re-collect a virus sample of a volume as low as 20 - 30 ml into the fluid
pathway using a designed sample applicator under operational conditions for
the manufacturing process ( flow rate 35 - 80 Lh-1, peak temperature 60 - 165oC)
at full scale. The complete pathway is disposable, hence offering an extraordinary
validation opportunity as well as a multi-product use and avoiding any potential
cross contamination.
Viral Clearance
Salmonella
There is a difference between virus inactivation and virus removal. Virus inactivation results in the irreversible loss of infectivity, while virus removal means the reduction of infective virus particles in number. The validation of viral clearance is very well described in regulatory guidelines, which also provides very clear recommendations on the choice of model viruses and the appropriate statistical approach for the calculation of the clearance factor.
With conventional heating methods, heat inactivation of virus is an attractive non-invasive physical treatment for high value pharmaceutical proteins. A drawback is the limited resistance of proteins exposed to elevated temperatures for times up to several hours. In addition, efficient heat transfer is often difficult in larger volumes. In order to achieve an inactivation process that allows for appropriate validation temperature gradients must be overcome. Microwave technology provides HTST heat treatment of product fluids in a continuous flow at flow rates up to 80 l/h. This allows for the inactivation of virus while maintaining the integrity of the pharmaceutical product.
By using microwave energy and a specially designed heat exchanger to heat and cool fluids faster than any known method - in milliseconds rather than minutes – the Dicam System achieves multi-log reduction of viruses and microorganisms in heat sensitive fluid materials such as biotechnically derived recombinant proteins, cell culture media, serum, plasma and plasma proteins. This multi-log reduction is accomplished without impairing the product's properties and without the limitations of irradiation, filtration or chemical methods. Delicate pharmaceuticals like egg-trays that cannot be autoclaved are particularly good candidates for microwave treatment.
Microwave heating allows for a rapid heat transfer in a continuous flow. Sensitive products such as pharmaceutical proteins are exposed for 180-680 milliseconds to high temperatures, hold times at peak temperatures are reduced to 2-4 ms. Heat denaturation of the protein molecules does not occur even at temperatures far beyond the melting temperature determined by differential scanning calorimetry. Protein precipitation is the first denaturing effect which can be observed: an appropriate selection of a respective carrier fluid (buffer) is demanding and might contribute significantly to expand the operational temperature range. Large protein molecules like immunoglobulins can be treated at temperatures which are destructive for small non-enveloped viruses, e.g. SV-40. Smaller protein molecules like tPA are stable to temperatures which inactivate the Parvovirus, which is highly heat resistant.
The superiority of HTST microwave heating becomes most evident in comparison to conventional virus inactivation: even at "moderate" temperatures (<80oC) which are applicable to heat sensitive proteins, a broad range of viruses including large non-enveloped viruses, e.g. Reo3, is fully inactivated (a range of viruses which is not accessible by solvent/detergent-, acid/pH3.0-3.9- or urea/3M- treatment).
Validation of Viral Clearance
The rationale for a validation of virus removal and inactivation is based upon possible hazardous side effects of potential viral contamination of a biotechnically manufactured protein product derived from mammalian cell culture, intended for use as a therapeutical drug or an in vivo diagnostic reagent.
Protein biosynthesis in mammalian cells features glycosylated proteins of high molecular weight with no disulphide bridge limitations and secretion of the protein as a singular, native molecule, which is folded correctly to a complex tertiar structure.
However, a potential retrovirus or adventitious virus contamination might lead to concerns on product safety. Even if no virus infectivity or reverse transcriptase activity can be detected for the master cell bank (MCB) or extended cell bank (ECB), the presence of virus-like-particles (VLP's) can often be demonstrated by electron microscopy . The microscopic evaluation gives no answer on the biological relevance of suspicious particles, especially regarding their infectivity; a prominent example is the presence of high numbers of A-type particles in hybridoma cells, where infectivity is extremely low or not detectable at all. Despite this discrepancy between the number of virus-like particles and infectivity, it is the general recommendation to calculate the overall reduction factor based on the particle number. This might induce the implementation of additional expensive but possibly unnecessary process steps.
Nevertheless, the presence of a virus of unknown origin cannot be excluded: an unidentified virus might have unknown and potentially harmful physiological effects, and it is the unknown nature of the virus contaminant which complicates the development of a specific assay. Without a specific and sensitive assay it is impossible to monitor the presence as well as the removal or inactivation of the virus along the downstream process of the protein drug.
With regard to a potential contamination by an unknown virus a number of preventive measures have to be established around and implemented into the complex manufacturing process of the protein in order to exclude the presence of any viral particle in the drug substance.
Preventive measures at the level of cell biology include extensive testing of the producer cells for specific viruses and testing for adventitious virus at a number of stages during fermentation.
The potency and efficacy of unit operations for the removal or inactivation of virus has to be demonstrated for the entire downstream process by the individual validation to those process steps that are considered to contribute to virus safety. Such validation has to be performed according to the very same concepts and criteria established for process validation in general.
With rare exceptions such as microwave induced High Temperature Short Time (•HTST•) heat treatment using the Dicam Microwaves System the scale of the respective validation equipment might be different from the manufacturing scale: Typically the validation needs to be performed using equipment at least of identical type and scale as intended for the manufacture.
Validation of viral clearance is different with that respect: the contamination of equipment for the production with infectious virus as a spike would impair such equipment to an irresponsible degree. In addition, the availability of the required amount of respective virus at high titer for a production scale up to several thousand liters is technically not feasible.
Red Mite
The British Free Range Egg Producers Association estimates that the annual cost of the red mite problem to the UK egg industry is in the region of £3.7 million. The biggest loss is believed to be from reduced egg production, costing UK producers nearly £2 million, while downgrading of eggs, bird mortality and the cost of insecticides and the labour to apply them each cost the industry over £500,000 a year.
© Dicam Microwaves APS