One of the main International Standards used as a reference document in a sterile pharmaceutical microbiology laboratory is ISO13408 – Aseptic Processing of Health Care Products .  Long ago, I reviewed the 1998 edition.  More recently, I acquired ISO 13208-1 (2008), so I compared my notes from the 1988 version with what is stated in the 2008 version.  The use of italics is my way of emphasising points.  Notes on changes are in blue.

INTRODUCTION

When I reviewed the 1998 edition, I did not note down any of the introduction.

• Anything labelled as sterile needs to made under tight control as part of a Quality Management System
• This part of the ISO focusses on the risks to the maintenance of sterility
• All possible sources of contamination need to be controlled and a risk based approach is recommended
• Appropriate validation of the aseptic process is needed and process simulation trials are an essential part of this

PART 1: GENERAL REQUIREMENTS

1. Scope

The ISO specifies the general requirements and offers guidance on the processes, programs, procedures, validation and control of the manufacturing process for sterile manufacture

3. Terms and definitions

Terms and definitions have moved to Section 3.

APA = Aseptic Processing Area

• ‘bioburden’ now defined as: population of viable microorganisms on or in a health care product and/or sterile barrier system
• ‘disinfectant’ now defined as: chemical agent that is able to reduce the number of viable microorganisms
  
• ‘media fill’ is defined as: method of evaluating an aseptic process using a microbial growth medium…media fills are understood to be synonymous to process simulation tests, simulated product fills, simulated filling operations, broth trials, broth fills, etc.” no longer defined
• ‘product sterilising filter’ is defined as: porous material with a nominal rating of less than or equal to 0.22mm, capable of retaining a defined number of microorganisms using defined challenge test and conditions. no longer defined
• ‘sterile’ is defined as “state of being free from viable microorganisms. NOTE: in practice, no such absolute statement regarding the absence of microorganisms can be proven”
• ‘sterilisation’ is defined as “validated process used to render a product free from viable microorganisms. NOTE: The number of microorganisms that survive a sterilisation process can be expressed in terms of probability. While the probability may be reduced to a very low number, it can never be reduced to zero.”

SECTION 3: QUALITY MANAGEMENT SYSTEMS  Now 4. Quality System Elements

• The Quality Management system is governed by the requirements in ISO 9001 and/or ISO 13485 Medical Devices – Quality Management Systems unless a superseding national, regional or international GMP is employed.

SECTION 5: FACILITY DESIGN  Now 6. Manufacturing Environment

6.1 General

6.1.1  Manufacutring environment should be designed and built in accordance with ISO 14644-4 (2001) Cleanrooms and associated controlled environments — Part 4: Design, construction and start-up

SECTION 12 MATERIALS AND EQUIPMENT DELIVERED TO ASEPTIC AREAS Now 6.4.2 Introduction of materials and components into the APA

Section 12.3 Bioburden

“The characterisation and resistance to inactivation of the bioburden on components and equipment that are introduced into the APA shall be periodically determined”.

Lots of reshuffling of information about in this section.  Either expands on the PIC/S recommendations, of they summarize this section.  Lots of great design information in this section. Just what one would expect.  From a VEM standpoint, pay particular attention to 6.6 Cleanroom qualification and 6.8 Environmental and personnel monitoring programs.  Loads of juicy information.

SECTION 7: SUPPORT AREAS OUTSIDE THE APA – Removed.  Seems to be incorporated into Section 6.8 Environmental and personnel monitoring programmes

• Section 7.1 6.8.1.6 The disinfection and environmental monitoring of this area (Class 100 000) may be less frequent than that utilised for the processing zones. Direct and indirect support zones may be monitored less frequently than the critical processing zone…
• Section 7.2 6.8.1.4  “Support areas shall be monitored routinely for the presence of microorganisms, i.e. environmental flora/isolates” The frequency of monitoring for the different zones shall be specified.

SECTION 14 ENVIRONMENTAL MONITORING PROGRAMMES Now 6.8 Environmental and personnel monitoring programmes

Section 14.2 Sampling

Section 14.2.1 Processing zones and frequency of sampling

• Section 14.2.1.1 page 14: “Product contact and material contact sites…monitored during and/or immediately after completion of the filling operation.” 6.8.1.5  The critical processing zone shall be monitored during each operational shift.  Monitoring approaches shall not compromise the sterility of a product.  NOTE Surface sampling is typically done at the end of the operation.
• Section 14.2.1.3 6.8.1.6  “Other processing zones…monitored at a defined frequency based on classification of the zones and testing data.”  Direct and indirect support zones may be monitored less frequently…
• Section 14.2.1.5 page 14: 6.8.1.4 (b)  “The frequency of such monitoring for different facilities shall be based on historical environmental monitoring data and the type of product and process.” …the frequency shall be based on historical environmental monitoring data and with consideration of regulatory requirements.

Section 14.2.2 Sampling sites

6.8.3.2 Sampling sites shall be selected based on a contamination risk assessment specific to a particular aseptic processing operation. Sampling sites should be derived from and consistent with those used during validation activities

SECTION 14.3 MICROBIOLOGICAL ENVIRONMENTAL MONITORING PROGRAMME Now incorporated into 6.8 Environmental and personnel; monitoring programmes
Section 14.3.1 General

• Section 14.3.1.1 page 14: “Periodic monitoring shall include methods for yeast, moulds and other microorganisms.”
• Section 14.3.1.2 page 14: “Validated recovery methods and calibrated equipment shall be used.”
• Section 14.3.1.4 page 14: “Additional microbial and particulate monitoring shall be performed following start-up of operations or following periods of extended shut-down or modifications to the facility.”
• The above is now nicely summarised in section 6.8.5 Monitoring procedures

Section 14.3.3 Microbiological sampling techniques

“Air sampling, using a volumetric sampling method, and other sampling methods, e.g. settle plates, swabs and contact plates, shall be used as appropriate to evaluate the microbiological quality of zones within the APA.”  Specifics are now in 6.8.3 Sampling for microbiological environmental monitoring

SECTION 15 ALERT AND ACTION LEVELS Now incorporated into 6.8 Environmental and personnel; monitoring programmes
Section 15.1 Development of alert and action levels  Now in 6.8.1.1 Alert and action levels and are derived from and consistent with results obtained from data trend analysis though the regs state what your limits should be

• Page 15 “NOTE1 Alert and action levels…derived from…results obtained during the aseptic process validation. Historical data…may also be appropriate..”
• Page 15 “NOTE 2 Often one level is appropriate…for critical processing zones.”
• Page 15 “NOTE 3 Adjustments of…levels could be appropriate, based (on)…media fill re-evaluations and…monitoring data.”

Section 15.2 6.8.6.2 Review of Data and trend analysis

Results need to be reviewed against established alert and action levels.  Impact of excursions needs to be assessed.

• “…Results of all environmental monitoring…reviewed in a timely manner against the alert and action levels…”

Section 15.3 Environmental monitoring trend analysis

• Section 15.3.1 6.8.6.2.2 “Environmental data shall be analysed for trends on a routine basis.”
• Section 15.3.2 6.8.6.3.1 “An investigation shall be initiated when necessary as indicated by trend data.”  Investigations carried out using documented procedures shall be initiated following events that indicate a possible loss of environmental control such as <details provided>
• SECTION 16 INVESTIGATIONS AND REPORTS Now incorporated into 6.8 Environmental and personnel monitoring programmes
  

• Section 16.1.1 page 15: “Investigations shall be conducted for unusual circumstances, extended mechanical breakdowns, or when action levels are exceeded.”
• Section 16.1.2 page 15: “Written investigation procedures shall be available and…include…data to be collected, extent of the problem, impact on product or environmental control, quarantine of the product, whether the environmental control has been attained, follow-up testing and notification of affected responsible personnel.”  Similar text is now in 6.8.6.3.1
  

Section 16.2 Investigation testing

• “Investigation testing shall be designed to locate the source of the problem and demonstrate that the area, process or equipment is once again under control. Revalidation may be necessary based on the outcome of the investigation.”  Statement removed.  Closest is 10.6.1.3 which says requalification should be performed where investigation identifies the need

Section 16.3 Investigation report

• Section 16.3.1 6.8.6.3.2 “The investigation shall be documented…”
• Section 16.3.2 page 16: “The report shall be written and approved by management..”  Statement to this effect in 6.8.6.3.2

SECTION 21.11 MICROBIOLOGICAL AND PARTICULATE ENVIRONMENTAL MONITORING  Incorporated into  Section 6.8 Environmental and personnel monitoring programs

• “A programme for microbiological monitoring of product transfer and freeze-drying shall be implemented.” Other contamination risks not specifically associated with aseptic processing are not addressed in this part of ISO 13408

SECTION 4: PERSONNEL Now 8. Personnel

• Section 4.1.2 Page 5: 8.1.2 The effectiveness of the defined documented procedures shall be evaluated at  intervals defined by the manufacturer defined intervals (with respect to personnel training).
• Section 4.2.2 8.2.2 point j) Page 5 discusses training of operators with reference to,”emergency procedures to protect product quality, eg. Loss of HVAC system, loss of power etc”.
• Section 4.2.3 Page 6 states “…personnel, who require temporary access to the APA (aseptic processing area) shall be accompanied at all times by a person trained and qualified in accordance with 4.2.2…”  Similar wording now in 8.2.6
• Section 4.2.5 Page 6 8.2.7 All personnel that directly participate in the filling of manufacture of sterile products in the critical processing zones shall have taken part in a media fill that meets the requirements of this part of ISA 13408 at least once per year.”
• Section 4.2.6 Page 6 states: 8.2.8  “new personnel…shall take part in at least one…media fill…before being permitted to participate in processes carried out in critical process zones.”
• Section 4.3 Page 6: General employee health notes: Initial and periodic medical examinations should be performed for individuals assigned to aseptic processing. 8.4.1 & 8.4.2 list general medical requirements.  The PIC/s GMP guide states something much like the above.
  
• Section 4.4 Page 7…”microbiological monitoring program…sampling of…garments and gloves.”  6.8.4.1  Personnel trained and qualified to work in the APA shall be subject to routine microbiological monitoring programme.  Monitoring data  Section 4.4.2 Page 7: “results of the (personnel) monitoring program shall be used to identify trends and evaluate the need for retraining.

SECTION 9: GOWNING Now 8.3 Gowning Procedures

Section 9.2 Gowning requirements

• Section 9.2.1 page 10: “Personnel shall wear dedicated factory clothing, including shoes, prior to entering the gowning area.
• Section 9.2.1 page 11 “NOTE 2 Personnel may change into dedicated factory clothing in the airlock adjacent to the gowning area.”
• Section 9.2.2 page 11 “NOTE A mesh hair net and beard cover, if required, are donned in the airlock. Disposable covers may be used in addition to dedicated shoes.
• Section 9.2.3 page 11 “NOTE 2 After a garment has been tested for microbial contamination (see 4.4), it should not be worn in the APA until cleaned.”
• Substantial changes to text.  Specifics about where to do what are not  stated.    I’d advise to stick to the above and  you should pay attention to notes 1-3 in section 8.3.2.3.

SECTION 13 PROCESSING TIME Now 9.2 Duration of the manufacturing process

• Section 13.1 page 13: “The compounding of bulk solutions shall be controlled in order to prevent potential increase in microbiological levels, and possibly endotoxins, that can occur up to the time that the bulk solutions are sterile filtered.”
• Section 13.2 page 13: 9.2 “The total time for the product filtration and filling operations, and holding time after filtration and prior to filling each unit operation of an aseptic process shall be minimized and  limited to a defined maximum.”

SECTION 10 CLEANING AND DISINFECTION OF THE APA Now spread through Sections 9.4 to 9.5

Section 10.1 Disinfectants and cleaning agents

The need to validate and document is emphasized.  Extensive changes to wording.

• Section 10.1.2 page 11: “Only cleaning agents and disinfecting agents which are validated and approved shall be used.”  No longer stated (as obvious?)
• Section 10.1.6 page 11: “The removal of disinfectant and cleaning agent residues on product contact surfaces shall be verified.”  9.5.2 Cleaning of equipment discusses

Section 10.2 Validation of disinfection procedures

• Section 10.2.2 page 12: “Each facility shall have appropriate procedures in place to evaluate, approve and control the use of disinfectants.” Refer to 9.5.3 Disinfection of equipment

Section 10.3 Monitoring effectiveness of cleaning and disinfection

• Section 10.3.3 page 12: “When unusual microbial results are encountered or persist, an investigation to identify the source of contamination shall be performed and documented.”   Now covered by 6.8 Environmental and personnel monitoring programmes

SECTION 17 MEDIA FILLS (PROCESS SIMULATION TESTS) Now Section 10. Process Simulation

Section 17.2 Initial performance qualification

Needs to cover  all parts of the aseptic process and include all aseptic manipulations.  Filter retentive capacity needs to be validated according to ISO 14208-2 and process simulation not intended to validate product (filter) sterilization.

 

• Section 17.5.1 page 18: “Media fill trials shall be conducted on separate days, at different times during the normal working period.” no longer stated.  presumably as long as all shifts and worst case batch size (max) is covered, revalidation is acceptable.
• Section 17.5.2 page 18: 10.3.1  “A list of permitted and proscribed interventions shall be prepared and retained  events…shall be available.”
• Section 17.5.3 page 18: 10.3.1 “Media fill trials shall be conducted…that include “worst-case” conditions…” …as far as is reasonably practicable, include permissible worst case conditions.
• Periodic requalification – media-fill acceptance criteria: if <500 unit per batch – 3 media fills. If ³500– 1 media fill.  Alert level = one contaminated unit in any unit.  Investigate and repeat run (³3000 units in batch, repeat run if values in Table 3 of ISO (located on page 21) are exceeded).  Action level = two contaminated unit in a single run.  Cease requalification, investigate and repeat initial qualification. (³3000 units in batch, cease, investigate and repeat if values in Table 3 of ISO (located on page 21) are exceeded).  Refer to Table 1 page 36 (10.9.2)

 

• Section 17.2.1 page 16: 10.5.1  “Performance qualification shall be conducted for each new aseptic processing operation  filling line and for each line and for each unique product new product/container configuration which that has not been represented in a previous performance qualification.
• Section 17.2.2 page 16: “Performance qualification…Representative criteria include: the actual product/container configuration…two products which bracket all others…products which have been deemed to be the worst case…”  See10.3.2 for new wording 
• Page 17: Initial performance qualification…acceptance criteria: minimum of 3 media fills. Alert level = one contaminated unit – investigate cause and conduct one additional run (and repeat it if it fails); Action level = two contaminated units in a single run, or one each in two runs – cease qualification, investigate cause, repeat initial qualification.  The number of simulation and the number of units filled per simulation are summarised in Table 1, as are the acceptance criteria.

Section 17.3 10.6 Periodic performance requalification

• Section 17.3.1 page 17: 10.6.1.1 “Scheduled…requalifications…shall be conducted twice annually at least every approximately every six months for each aseptic process and product/container configuration and aseptic filling line.”

 Section 17.4 10.7  Repeat of initial performance qualification

• Section 17.4.2 page 18: “An aseptic process of filling line shall be subject to repeat performance qualification studies when:
  • requilification has failed an action level is exceeded
  • production lines have not been in operation for an extended peri
  • there has been a significant change
• Section 17.3.3 page 17:  10.9.1 “Product manufacturing may resume while the media-filled units are being incubated; however, product release shall not occur until acceptable media-fill data are obtained.” All product that has been produced on a line subsequent to the process simulation shall be quarantined until a successful resolution of the process simulation has occurred.Section 17.5 10.3 Media-fill Simulation procedures

Section 17.6 10.2 Media selection and growth support <<up to here>>

Section 17.6.3 page 19:  10.2.1 “The microbiological growth  media selected for process simulation media-fill runs shall be capable of growing a designated group of reference wide spectrum of microorganisms and of supporting microbial recovery and growth of low numbers of microorganisms, i.e. 100 colony forming units (CFU)/unit or less.

Section 17.6.1 page 19: 10.2.3 “Verification of growth promotion media used in specific media-fill runs shall be conducted following the run.”

Section 17.6.2 page 19: “The incubation temperature shall be the same as that used for the media-filled units.” No longer stated, but the logical incubation temperature.

Section 17.7 10.4 Incubation and inspection of media-filled units

• Section 17.7.1 page 19: 10.4.2  “Units that are leaking, broken or otherwise damaged media-fill evaluation units to the extent that there is no question of their not being rejected during routine documented visual checking shall be recorded and removed, and a record made of such removal, following processing and prior to incubation of the media.”
• Section 17.6.2 page 19:  10.4.3 “Media-filled units shall be incubated for not less than a minimum of 14 days.”  Temp range stated here.
• Section 17.6.3 page 19: “Incubation temperatures shall be appropriate for the specific growth requirements of microorganisms that are anticipated in the aseptic filling area.”
• Section 17.7.4 page 19: 10.4.1 “Media-filled containers units shall be agitated, swirled or inverted before incubation to ensure stored or manipulated to allow contact of the media with all product contact surfaces in the unit.”
  interior surfaces of the container.
  
• Section 17.7.5 page 19: 10.4.4 “After…incubation period…media-filled containers…visually inspected for…microbial growth.” Now says to inspect via defined procedure.
• Section 17.7.6 page 19: 10.4.5 “Microorganisms present in…units shall be identified…”
• “NOTE 1 Inspection of the units at an earlier time period (3d to 7d incubation) can be useful to gain a preliminary indication of the results.”
• “NOTE 2 Media-filled units should be chronologically identified within the batch…”  interestingly, no longer stated.  Knowing at what point during the fill the contamination occurred is valuable information when investigating a process simulation failure as it could point to a specific simulated intervention.  With that knowledge, one can examine batch records for the intervention and determine batch disposition of what may or may not be released batches (since last media fill).  Nope, missed it.  It’s in section 10.3.7.

Section 17.11 10.5.3 Media-fill runs exceeding action levels Acceptance Criteria

Section 17.11.1 Investigation

• Section 17.11.1.1 page 22: “When…action levels… exceeded… investigation… conducted and documented…”
• Section 17.11.1.2 page 22: “…action levels…exceeded…prompt review of all appropriate records relating to aseptic production between the current media fill and the last successful one.
• 10.5.3.2 Any contaminated  unit shall result in an investigation to determine cause (if possible)

“The investigation should include, but not be limited to, consideration of the following: no longer stated, though handy to make note of.

1. Microbial environmental monitoring data
2. Particulate monitoring data
3. Personnel monitoring data (finger impressions, etc)
4. Sterilisation cycles for media, commodities and equipment
5. HEPA filter evaluation
6. Room air flow patterns and pressures
7. Operator technique and training
8. Unusual events that occurred during the media fill
9. Storage conditions of sterile commodities
10. Identification of contaminants as a clue to the source of the contamination
11. Housekeeping procedures and training
12. Calibration of sterilisation equipment
13. Pre and post-filter integrity test data, and/or filter housing assembly
14. Product and/or process defects, and/or limitation of inspectional processes; and
15. Documented disqualification of samples for obvious reasons prior to final reading

Section 17.9 Contamination with media

“Contamination of the facility and equipment with media during the media-fill runs shall not compromise…the facility and equipment or the product…using the same facility and equipment.”  No longer be stated.  Probably as cleaning procedures and line clearances should be validated and the process documented, so spilling growth media in a filling room will have a cleanup procedure, and you could always conduct a “VEM full screen” of a room after completion and cleanup of a process simulation.

Section 17.10 10.8 Documentation of process simulations Data required for media fills

“All process simulation media-fill runs shall be fully documented, and the following information included…:

Words to this effect.  Media fill replaced with process simulation; filling room with processing area, etc.  A- Q, not numbered.

1. date and time of media fill
2. identification of filling room used
3. container/closure type and size
4. volume filled per container
5. filling speed
6. filter lot and catalogue number no longer stated – would be in batch record as everything needs to be documented
7. type of media filled
8. number of units filled
9. number of units rejected at inspection and reason
10. number of units incubated
11. number of units positive
12. incubation time and temperature for each group of units incubated and whether any group of units is subjected to two different temperatures during the incubation
13. procedures used to simulate any steps of a normal production fill, which might include, for example, mock freeze-drying or substitution of vial headspace gas
14. microbiological monitoring data obtained during the media-fill set-up and run
15. list of personnel who participated in the media-fill
16. growth promotion results of the media removed from filled containers
17. identification of the microorganisms from any positive units, and investigation of any contamination events observed during media fills split into two points
18. management review
19. length of time media was stored in holding tank prior to filtration no longer stated – would be in batch record as everything needs to be documented
20. length of time taken to fill all containers no longer stated – would be in batch record as everything needs to be documented

SECTION 18 FINISHED-PRODUCT STERILITY TESTING Now Section 11. Test For Sterility

Section 18.1 General

• Page 23: 11.1 General “Where Sterility testing is required for of aseptically filled products this testing shall be conducted for each batch or lot.”
• Section 18.2.1 page 23: 11.2.1 “Positive units from a test for sterility sterility-test results shall be evaluated and an investigation shall be initiated…”
• Section 18.2.2 page 23: 11.2.2 “A correlation assessment between types of microorganisms found in the manufacturing environment, sterility testing room, and isolated from failed sterility tests shall be conducted.” says pretty much the same thing, though words moved around.

Section 18.3 Sampling Plans

• Section 18.3.1 page 23: “Sampling plans…developed to assure representation of the entire batch…”

SECTION 19 STEAM IN PLACE  Appears to have been removed entirely.  Part 5 of ISO 13408 details Sterilization in place.

Some discussions on sterilisation using various methods is provided in Section 9. Manufacture of product

Section 19.1 General

• Section 19.1.1 page 23: “Equipment which cannot be sterilized in an autoclave…shall be steam sterilised in situ with a demonstrated process lethality of six logarithms reduction…”
• Section 19.1.2 page 24: “In the absence of an automated SIP system, manual procedures shall be defined, adhered to, and documented.”

SECTION 20 PROCESS FILTRATION  Appears to have been removed entirely. Part 2 of ISO 13408 details filtration.

Section 20.1 Filter and filter equipment evaluation programme

• Section 20.1.1 page 24: “A documented filter evaluation programme shall be established prior to validation and acceptance of filters.”
  • “The shedding of media-migration characteristics of the filter should be evaluated based on the intended life of the filter.”
• Section 20.1.2 page 24: “Product sterilizing filters shall be evaluated by an appropriate and defined challenge test, or evidence of product-specific data from the filter manufacturer shall be available.”
• Section 20.1.3 page 24: “Absorption or adsorption the product by the filter shall be evaluated.”

Section 20.1.1 Biological Testing

• “If any plastics are used as components of filters/filter systems, biological safety testing should be conducted on each plastic material.  Testing may be conducted on the total filter/filter system.  Filters should be evaluated for the presence of endotoxins.”

Section 20.4 Filtration in the filling line

• Section 20.4.3 page 25: “The filter manufacturer’s lot number shall be included in batch manufacturing records.”

SECTION 21 FREEZE-DRYING Removed entirely.

Section 21.4 Process Routing

• ”Facility design related to location of equipment for filling, freeze-drying and sealing shall be such that transport of open product is kept to the minimum possible.”

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