Production Process

The following schematic flow chart is an illustration of the process adopted to produce SPF Embryonated Eggs. The SPF genetic material is also regenerated by a full term incubation that is undertaken every 14 to 16 weeks.


The critical steps and processes deployed for the production of SPF embryos include the following:

Propagation and regeneration of SPF Genetic Stock

An SPF genetic embryo (Fertilized SPF Egg - SPFE) free from the listed pathogens for a minimum of three generations is the most logical starting point of the cyclic production process. These embryos are incubated in automated Primary Incubators (Setters) that maintain a temperature of 99.5oF, and predetermined levels of RH and CO2 and are supplied with sterile HEPA filtered air. The SPFEs are automatically rotated every hour during the entire incubation process to prevent the zygote to adhere to the shell. At the conclusion of 18 days the SPFEs are removed from the Setters and candled aseptically. Underdeveloped and dead embryos are discarded. The candled SPFEs are transferred to a secondary incubator (Hatcher) that is similar to the first incubator with the exception that it does not turn the eggs and the temperatures, RH and CO2 levels differ. The SPF stock hatches out in 3 days post transfer or in 21 days of total incubation. Each SPF chick is identified by a unique number and transferred to stalls where they are maintained till 8 weeks of age at specified temperatures. At 8 weeks the SPF pullets along with 20% males are selected and the balance either liquidated or transferred to isolators for the manufacture of diagnostic reagents. The SPF adolescent stock, commonly referred to as growers, is maintained in the same stall for an additional 8 weeks at the conclusion of which they are transferred to adult stalls (age of transfer). The SPF males are reduced to 10% and the balance liquidated or transferred to isolators for the manufacture of diagnostic reagents. SPF Embryonated eggs are collected from the 25th week of age by which time the size of the embryonated egg and the fertility has reached optimal levels.The SPF stock remains in the adult stall till liquidation.

Personnel Entry and Automated Systems

Personnel are considered to be potentially the most hazardous and the highest source of sterility breakdown. All personnel are medically examined at pre-determined intervals. Upon arrival at the facility these personnel are quarantined. Personnel entry into the work area is limited to authorized personnel only. Detailed protocols control the process. In the first instance personnel will shower in through a three change room process. Personnel from one unit will not enter the second unit. Personnel entry into the SPF stalls is limited to daily inspection. Activities are monitored and recorded by a CCTV. Each stall has an adjacent anteroom from where nutrients are loaded into the automated systems and SPF Embryonated eggs removed. All operations are carried out by automated systems. The SPF stock is fed nutrients and water using automated systems. The stock is housed on removable plastic slats that facilitate cleaning. SPFEs are collected using an automated roll-away system that is connected to a conveyor system. The entire process is monitored through a remote CCTV system to allow for a minimum human intervention. This reduces the chances of contamination through human contact.

SPF Monitoring

Blood and other samples are collected at weekly and monthly intervals to check for any evidence of the specified pathogens in the stock.  All birds are tested for the range of pathogens listed in the Pharmacopoeia at the intervals and levels specified in the Pharmacopoeia. These include a list of 26 pathogens that include Avian Influenza Type A. 

A well equipped laboratory is an integral requirement of an SPF facility and employs serological and microbiological methods to examine the samples to ascertain and certify a specified pathogen free status of the stock.

International Pharmacopoeia requirements stipulate that once a source SPF flock is defined, no non-SPF birds can be added to it.

Treatment of Air

Ordinarily reference to an “environmentally controlled building” would imply an air conditioned building which may or may not be a process requirement. However, in the specific instance of production of SPFE, it is a critical process requirement without which SPFE production cannot be undertaken.

Ambient air is filtered through a series of filters and finally through High Efficiency Particulate Air (HEPA) Filters that ensure particulate filtration to a level of 0.22 microns in the work space. The HEPA filtered air quantity varies between 2400 M3 to 8640 M3 per hour per stall dependent upon body mass air temperature and RH. Air streams of each stall are independent to prevent cross contamination. In totality this translates to 14,400 M3 to 51,840 M3 of HEPA filtered air per hour. No air is re-circulated and 100% of the filtered air is exhausted. The air exhaust system runs independent of the air supply system and is suitably designed to prevent suck-back of unfiltered air in the event of power outage or drop in pressure. The air velocity is maintained at 1.2 m/s at the inlet into the rooms.

Work space is maintained within a specified temperature range of 18 deg C to 25 deg C The working space is subdivided into several zones each maintained at a differential air pressure of 20 - 30 Pascal in critical areas and graded down to 10 Pascal in relatively lower critical areas. Air velocity, RH and Ammonia are the other physical parameters that require control. The interplay of air temperature, air velocity, humidity and air quantity is monitored and dynamically controlled by a sophisticated PLC enabled Building Management System. 

Raw Material processing and treatment

All raw materials used for the SPF stock are meticulously screened. No raw material from animal sources is used. Feed is ground in a hammer mill and mixed in standard mixers or extruded/extended outside of the facility. Feed is packed in double lined packages and the outside layer is removed at the facility. Material is passed into the facility through a decontamination chamber where it is subjected to fumigation and neutralization. No un fumigated material is allowed into the facility. Smaller sterilizable material is steam sterilized in autoclaves. This treatment insures that no external pathogen gains entry into the process.

The water used for the genetic stock requires similar treatment. It is pre-filtered, softened, deionised and then terminally sterilized using either chemical or physical methods.

SPF monitoring

The product is collected from each stall, candled, graded, and stored in walk-in cold storages. They are marked for identification and traceability purposes and SPFEs that meet specifications are aseptically packaged into decontaminated cartons and stored / transported in refrigerated transport vehicles.