MICLAB-075 Micro Evaluation on Bioburden, Non sterile and Raw Materials

Correct Aseptic technique must be used when performing any Microbiological procedures.

Safety glasses and gloves are to be worn when using IPA/Solvent.

All testing must be preformed within a LAF cabinet.

1. Equipment preparation For Non-sterile testing:

1.1               Assemble filtration units together.

1.2               Place 6 filtration units in a dacron bag, place autoclave tape with Date Autoclaved on the tied section.

1.3               Place 6 forceps inside individual test tubes, then place the 6 filled test tubes inside a Sterilope bags, seal by folding over twice and applying autoclave tape.  Add the Number and Item and Date Autoclaved on the front of the bag.

1.4               Follow the above procedure for preparation of spoons and scissors.

1.5               Place the wrapped bags of forceps, scissors and spoons into the wire rack for the autoclave.

1.6               Autoclave the items in laboratory autoclave.

2. Procedure

2.1               Non‑sterile products and raw materials are tested to determine the presence of (Presence Test) and/or number of (Limit Test) micro‑organisms present.  The type of organisms present may also need to be evaluated.
Some common pathogenic organisms must be absent (refer to Specifications).  The potential pathogenicity of other Gram Negative rod organisms is to be evaluated in the particular situation and a maximum permissible level for other organisms is set.

2.2               Incubation Temperatures and Times for media used in the testing of Non‑Sterile Products and Raw Materials are as follows unless stated differently in the individual product’s control method.

3. Bulk Solution Bioburden (BSB) Samples

3.1               Sampling (See MICLAB 095)

All batches manufactured are to be sampled for Bulk Solution Bioburden (BSB) testing.

Production personnel are responsible for ensuring that a BSB sample is taken for every batch manufactured.  The Bioburden sample is collected at the conclusion of the filling of the batch prior to filtration of the solution.  Operators aseptically disconnect the hose from the filter assembly and aseptically fill one sterile 500mL bottle.

These samples include:

1)    Excipient sample

2)    Polysorbate sample

3)    Wash water sample.

3.1.1         Testing:

Filter 100mL of the Bioburden sample through a 0.45µ membrane filter, rinse with 150mL Peptone Water.  Place the whole filter onto an R2A plate and incubate at 30-35°C.  Examine after 5 days.
Write the date of testing on the BSB label and refrigerate the remaining sample.

3.1.2         Results:

If count obtained is over the Alert Level of 100 and less than 200 colonies, conduct a Gram stain on different types of colonies present.

If count obtained is over 200 colonies and not countable a retest is to be performed.  Gram stain is required on the original sample, which is over the Alert Level.

If any Gram negative rods are present, identify for conformance.  Record all results in log book.
If a retest is required, apply the same test method to the original sample using 10mL and 1mL volumes so that an exact count is obtained.

If a re-retest is required apply the same test method to the original sample using volume less than 1ml, i.e. 0.5ml and 0.1ml as to obtain a count.

After reading and recording the result in the log book, the BSB bottles can be cleaned out of the refrigerator,

OR either retested.

Every 6 months, graphical representations of Bioburden results are to be printed and stored in the Bioburden Reports File.

If the Bioburden is more than alert level, inform the Microbiology Manager who will decide if any additional testing of the product is required, e.g. LAL Pyrogen test.  The reason for release is to be added in the ‘Comments’ section of the log book.

4. Filled Container Bioburdens (FCB)

4.1               Example-Prior to Autoclaving

All batches manufactured are to be sampled for Filled Container Bioburden testing.

4.1.1         Predetermined units are to be randomly selected immediately prior to the autoclaving of the final load.  These samples are to be labelled “Bioburden” and placed into a plastic bag and delivered to the Micro. Lab. (see MICLAB 095).

4.1.2         Aseptically pool specified amount from each of the units into a sterile Membrane Filtration unit and filter through a 0.45µ membrane filter and wash with sterile Peptone Water.  Transfer the filter into a sterile petri-dish containing an absorbent filter pad moistened with Tryptone Soy Broth.

4.1.3         Incubate at 30-35°C for 5 days under humid conditions.  If any colonies are present Gram stain & record the morphological and microscopic appearance. If any Gram-positive or negative rods are present, identify further.

4.1.4         Record the results in the log book. Specify Alert and action limits.

5. Raw Material Bioburdens, (RMB)

Sterile operator’s sample raw material for microbiological analysis and the samples are either delivered directly to the laboratory or placed into the micro sample cabinet within production.

6. Surgical Face Masks

Every batch of Face Masks purchased must be examined for microbiological status prior to use.  On delivery of every batch, 5 boxes of facemasks will be randomly sampled by the Warehouse Sampling staff, and delivered to the Microbiology Laboratory. Test as per Raw Material Specification and test method.

7. Isopropyl Alcohol (70% IPA)

Isopropyl Alcohol is routinely used throughout the Microbiology Laboratory.  The Production Services team prepares the 70% IPA.  A sample from each new keg is to be aseptically sampled into a sterile 100ml bottle and sent to the Micro Lab for testing,

The following method is used for IPA samples and all growth is to be evaluated:

7.1               Samples must be collected in sterile 100mL bottles.  Filter the sample (100mL) through a sterile 0.45µ Hydrophobic Membrane filter.  Wash the filter with 150mL of sterile Peptone Water.  Aseptically transfer the filter into a 100mL bottle containing 100mL of sterile Letheen Broth.

7.2               Incubate bottle at 32°C for 5 days.  If growth is observed, subculture onto a Nutrient agar plate and incubate for 48 hours at 32°C.  All colonies are to be gram stained.

7.3               Record the results in appropriate log book.

Note: When entering IPA batches into record book, a batch number must be allocated.

Alert Level for prepared IPA: Zero organisms/100mL.

8. Speciation Procedures for Organisms found in Non‑Sterile Products and Raw Materials

8.1               If after incubation micro‑organisms are apparent in either the bottle/s of medium or Agar plates for any Non‑Sterile Product or Raw Material tested for the Presence of Micro‑organisms and/or Microbial Limit Test, the following steps should be carried out:

a) The fact that growth has occurred and in which medium and the time period involved, and the number of colonies present, should be recorded into the appropriate Recording System.

If further ID is required, according to the control or raw material method specifications:

b) The organism/s present is/are to be streaked out (for isolated colonies) onto a Nutrient Agar plate and incubated at 32°C for 48 hours.  If growth has occurred in the FTM also streak the contaminant onto Reinforced Clostridial Agar plate and incubate at 32°C in an anaerobic jar for 48 hours.

c) A Gram stain of the different organisms present must be carried out and details of their microscopic and macroscopic appearance recorded appropriate Recording System.

d) A retest on the product may have to be carried out ‑ details are given in Section 10, “Retest Procedures”

e) If the following organisms are found, take the action detailed below:

8.1.1         Gram Positive rods or Moulds
Take no action unless they are in excess of the Action Level specified for each product or Raw Material or they are shown on retest to have increased in number.

8.1.2         Gram Negative cocci

Inform the Microbiology Manager if GNC found in product. Further investigation and Identification is required, including a KOH test to confirm that the organism is Gram negative.

8.1.3         Yeast
The requirement is for absence of Candida albicans.  If the yeast is shown to be other than Candida albicans take no action unless they are in excess of the Action Level specified for each product or Raw Material or they are shown on retest to have increased in number.

Note: See MICLAB 070 for Identification of Candida albicans.

8.1.4         Gram Positive Cocci
The requirement is for absence of Staphylococcus aureus.  Heavily streak the suspected organism onto a Mannitol Salt Agar plate and incubate at 32°C for growth for a maximum of 3 days.  At the same time streak the suspected organism onto a Nutrient Agar plate and incubate at 32°C for 48 hours.  (In the case of needing to perform the test.)  Presumptive coagulase – positive Staphylococci produce colonies with bright Yellow zones, while (non-pathogenic) coagulase-negative Staphylococci are surrounded by a Red or Purple zone.

Presumptive Staphylococci aureus must be confirmed with a test (coagulase test).  Use the colonies grown on the Nutrient Agar plate to perform this test. Speciation of S. aureus based on:

Gram stain ‑ Gram Positive cocci in clusters.

Catalase reaction ‑ Positive.

If no colonies are observed on the MSA plate the organism isolated can be considered to be other than S. aureus.  Assuming correct incubation conditions have been met.

If the organism is shown to be other than S. aureus take no action unless they are in excess of the Action Level specified for each product or Raw Material or they are shown on retest to have increased in number.

Note: microorganism Identification System (eg VITEK 32) can be used for the further identification of Gram Positive Cocci, if required.

8.1.5         Gram Negative Rods
The requirement is for absence of Salmonella sp., Pseudomonas aeruginosa (and all Pseudomonads including Brevundimonas, Burkholderia, Sphingomonas, Stenotrophomonas and Xanthomonas), Escherichia coli and any other potentially pathogenic Gram Negative rod.  The potential pathogenicity of the isolated organisms is to be evaluated in the particular situation i.e. with regard to the product type.

8.1.6         If any Gram Negative, oxidase negative rods are present they are to be identified further.
If the organism is a Salmonella it will be necessary to have the organisms serotyped at a Reference Laboratory.

8.1.6.1       If any Gram Negative, oxidase positive rods are present, identify further.

8.1.6.2       If the organism is shown to be other than a Salmonella, E. coli or Ps. Aeruginosa (and all Pseudomonads including Brevundimonas, Burkholderia, Sphingomonas, Stenotorphomonas and Xanthomonas) or in the particular situation it is not considered to be a potential pathogen, take no action unless they are in excess of the Action Level specified for each product or Raw Material or they are shown on retest to have increased in number.

9. Out-of-Specification Procedures for Non‑Sterile Products and Raw Materials

9.1               General

9.1.1           In the initial instance of recording an OOS result the manager present must be informed and a Deviation Report (DR) may be required.

9.1.2           A test may be repeated when the Initial test has been invalidated due to an error in procedure, (including equipment, Technician, etc.).

9.1.3         All OOS results will be reviewed in a two-phase approach.  Phase 1 is the review of the testing procedures by the microbiology technician to eliminate the possibility of lab introduced error or contamination.  Phase 2 will investigate the OOS result in more detail.

9.1.4         Under normal conditions Alert Level excursions will only require organism identification and simple trend reports, however, for Action Level excursions it is necessary to investigate thoroughly to determine a definitive cause and recommend corrective and preventative actions, (CAPA).

9.1.5         It is recommended that certain aspects of the investigation involving review of data held by other departments is delegated to a representative of the relevant department.

9.2               Phase 1 and 2 Investigation Responses

9.2.1         Phase 1 investigations for Non-Sterile and Raw Material OOS results must consider, (but are not limited to), the following details:

1. Correct collection, storage and preparation of test sample and collection vessel.

2. Correct set-up of test equipment.

3. Correct method used for test.

4. Completed Training records.

5. Expiry of consumables and equipment observed.

6. Media stasis satisfactory.

7. Correct incubation conditions of test.

8. Observations made during test procedure.

9. All calculations checked.

10. All test control results, where relevant, are satisfactory.

11. Review of other test results for the test session, to determine a trend.

12. The above items must be clearly addressed in the DR for OOS results

9.2.2         Phase 2 investigations involve a thorough systematic analysis of the incident, to determine the root cause for the OOS result. Phase 2 investigations are to be tailored for each incident to reflect the significance of meeting or exceeding either the ALERT or ACTION Levels.

9.3               Phase 2 Investigation Responses

9.3.1         Determine if the alert excursion is to become an action level excursion that requires upgrading the notification to an action level based on the following criteria:

9.3.2         Three alert/action level excursions from the same sterile area on the same day.

10.3.3      The minimum requirements for most situations are listed as below, this list is by no means exhaustive and specialist assistance from departments other than Microbiology should be utilized in the investigation.

10.3.4      All affected departments will perform their part of the investigation, carry out corrective actions and findings of the investigation.

9.4               OOS Bulk Solution Bioburden

9.4.1         Alert Level

Results obtained over the alert level require the following actions

Gram stain

Further identification as outlined in section 9 ‘Specification Procedures for Organisms found in Non-Sterile Products and Raw Materials’

9.4.2         Action Level (In addition to alert level requirements)

Results obtained over the action Level require the following details

Results of any material used to manufacture the batch e.g. WFI & raw materials

Sterilisation of vessels (SIP)

Cleaning records for vessels (CIP)

Review of other test results to determine a trend.

Environmental conditions conform

CAPA recommendations

9.5               OOS Filled Container Bioburden

9.5.1         Alert Level

Results obtained over the alert level require the following actions

Gram stain

Further identification as outlined in section 9 ‘Specification Procedures for Organisms found in Non-Sterile Products and Raw Materials’

D-values where applicable.

9.5.2         Action Level (In addition to alert level requirements)

Results obtained over the action Level require the following details:

Results of any materials used to manufacture the batch e.g. WFI & raw materials

Sterilisation of vessels (SIP)

Cleaning records for vessels (CIP)

Packaging bioburden

Environmental conditions conform

Incidents inside sterile

Review of other test results to determine a trend.

CAPA recommendations

9.6               OOS Raw Material Bioburden

9.6.1         Alert Level

Results obtained over the alert level require the following actions:

Gram stain

Further identification as outlined in section 9 ‘Specification Procedures for Organisms found in Non-Sterile Products and Raw Materials’

D-values where applicable.

9.6.2         Action Level (In addition to alert level requirements)

Results obtained over the action Level require the following details:

Environmental conditions where sampled.

Review of other test results to determine a trend.

Contact supplier.

Investigation meeting.

CAPA recommendations.

9.7               OOS Non-Sterile

9.7.1         Alert Level

Results obtained over the alert level require the following actions:

Gram stain

Further identification as outlined in section 9 ‘Specification Procedures for Organisms found in Non-Sterile Products and Raw Materials’

9.7.2         Action Level (In addition to alert level requirements)

Results obtained over the action Level require the following details;

Results of any material used to manufacture the batch e.g. WFI & raw materials.

Sterilisation of vessels.

Cleaning records for vessels.

Machine sterilisation charts.

Review of other test results to determine a trend.

Environmental conditions conform

CAPA recommendations

9.8               OOS Mask Bioburden

9.8.1         Alert Level

Gram stain

Further identification as outlined in section 9 ‘Specification Procedures for Organisms found in Non-Sterile Products and Raw Materials’.

9.8.2         Action Level (In addition to alert level requirements)

Results obtained over the action Level require the following details

Additional testing may be required.

Put masks on hold and hold an investigation meeting.

Contact supplier.

9.9               OOS IPA

9.9.1         Alert Level

Gram stain

Further identification as outlined in section 9 ‘Specification Procedures for Organisms found in Non-Sterile Products and Raw Materials’

9.9.2         Action Level (In addition to alert level requirements)

Results obtained over the action Level require the following details

Additional testing may be required.

Contact immediately to ensure IPA is not distributed to production.

Contact immediately to resample the batch.

10. Retest and Repeat Procedures for Non-Sterile Products and Raw Materials

10.1            Repeat Procedures

A test may be repeated when the initial test has been invalidated due to an error in the procedure (including equipment, Technician, etc).

10.2            Retest Procedures

Where the number of micro‑organisms in the Non‑sterile product or Raw Material detected in the Microbial Limit Test is over the ALERT LEVEL for that material, a retest on the Non‑Sterile product or Raw Material in question should be carried out.  The retest involves repeating the Microbial Limit Test in triplicate, for both Limit and Presence testing.  If after retesting the product or raw material three times the number of micro-organisms is still over the ALERT LEVEL the situation is to be evaluated by the Microbiology Manager.
Note:
If there is not a sufficient quantity remaining from the original sample for either repeat test or the re-test, new samples should be acquired by randomly sampling throughout the batch.

11. Summary of Changes