Aseptic manipulations

Grade A is the highest standard, and is required for local zones where high-risk tasks are carried out. Examples are the aseptic filling of a product following filtration through a 0.22 pm filter to render it sterile, and general aseptic manipulations. This is usually achieved by the use of cabinets or hoods that enable laminar airfiow patterns to be established. 

One of the most potent routes for transmission of bacterial disease is via the air. Cross-infeetion in hospital wards, infeetion in operating theatres, the transmission of disease in elosed spaces such as cinemas and other places of assembly, in the ward rooms and erew s quarters of ships and in submarines are all well known. Of equal importance is the provision of a bacteria-fiee environment for aseptic manipulations generally. Clearly, the disinfeetion of atmospheres is a worthwhile field of study and to this end much research has been done. It is equally clearly important to be able to evaluate preparations claimed to be air disinfeetants. 

Aseptic manipulations should be performed in the sterile air of a laminar airflow unit. Speed, accuracy and simplicity of movement, in accordance with a complete understanding of what is required, are essential features of a good aseptic technique. 

Once the medium has been processed, it is held for a period of time at least equal to that for aseptically produced materials. Any aseptic manipulations performed during and at the end of the hold period should be simulated as well (i.e., sampling, refiltration, hold times, and product recalculation). 

Any of aseptic manipulations performed during and at the end of the hold period should be simulated hold times and product recalculation. 

Validation of the lyophilizer cleaning and sterilization processes should be accomplished. Particular care should be taken to verify that there is no back-migration of contaminants, whether from adjuvant fluids integral to the equipment of by cross-contamination from previous product. Typically, an overkill approach using a sufficient number of thermocouples and biological indicators is the method of choice. Finally, fill testing to verify the adequacy of the sterilization procedure and the aseptic manipulations involved with product filling, transfers, and lyophilization needs to be performed. 

Adherent insect cell release from solid surfaces generally does not require trypsinization, unlike anchorage-dependent mammalian cells. Insect cells, like mammalian cells, need rigorous aseptic manipulation during cell transfer, inoculation, and propagation in bioreactors. A minimal inoculum density is required for both cell types. Typically, insect and mammalian cell cultures are initiated with inocula of 1-2 X 105 cells per milliliter of liquid medium (Agathos, 1991). Table 2.3 summarizes the major differences and similarities between these cells. 

Horizontal-flow cabinets are satisfactory for aseptic manipulations such as media preparation where potentially hazardous aerosols are not generated. In these the filtered air enters through the back of the cabinet and leaves through the front. A horizontal-flow cabinet is a miniaseptic room which is much easier and cheaper to maintain than a walk-in aseptic room. In addition, it has the advantage that the worker is not confined within a small room which may become extremely hot and stuffy and, secondly, that the worker s head is separated from the cultures by a perspex screen thus further reducing the chance of contamination. 

Prior to introduction of the working materials into the LAFD, the gloved hands of the operator should be thoroughly washed and rinsed to remove dry lubricants, sanitized by spraying with 70% alcohol, and allowed to dry in the laminar airstream. The working materials may then be transferred to the work zone, and aseptic manipulations begun. The operator s... 

Aseptic manipulations must be carried out in the grade A air of a laminar flow cabinet. Speed, accuracy and economy of movement are essential features of good aseptic technique. It is therefore essential that workers are well trained and motivated and familiar with the task in hand. Observation and microbiological monitoring of the operator and of the environment are very important. Air quality is measured using settle plates or slit samplers, work surfaces by taking swabs or by use of contact plates (Chapter 20). 

The USP describes two general methods for conducting the test the direct transfer, or direct inoculation, method and the membrane filtration method. As the name indicates, the direct inoculation method involves the aseptic transfer of a sample of test product solution into the sterility test growth medium. To use this method, it must first be demonstrated that the product solution itself does not inhibit the growth of typical indicator microorganisms specified in the USP method. It should be self-evident why it is important to perform testing to negate the chance of product inhibition of possible microbial contaminants, as this is the purpose of the sterility test. The direct inoculation method, while not theoretically complex, requires the utmost technical precision and aseptic manipulation techniques for proper execution. As a consequence of the repetitive motions involved, it is prone to human error. 

Within the filling room, all aseptic manipulations and process stages requiring exposure of the dosage form or product contact components should be protected by localized laminar flow drawing air from the Class 100 environment. The importance of this protection is not in the sterility of the air but in the sweeping effect of the air. 

Rooms for aseptic processing should, in the unmanned state, comply with the conditions specified for Grade 1/B in the Table of Basic Environmental Standards (Appendix 1). With people present and work in progress, Grade 1/A (Appendix 1 conditions should be maintained under the contained work stations where products are exposed and aseptic manipulations carried-out. 

Sterilization in place—Closed systems such as process vessels, dryers, centrifuges, isolators, and other items should be subjected to a validated sterilization procedure, which assures that all internal surfaces have been rendered free of microorganisms. Sterilization-in-place (SIP) procedures reduce the number of aseptic manipulations necessary to ready the equipment for use in the aseptic production processes and as such are considered preferable to aseptic assembly of systems from individually sterilized components (25). The SIP procedure should allow the system to maintain sterility until ready for use without aseptic manipulations. Sterilization-in-place procedures could employ steam, gas, dry heat, radiation, chemical agents, or other validateable sterilization procedure. 

---