Verification Protocol

Protocol Reference:

2Contents 1. About BioBall Multishot 550 32. About This Protocol 43. Safety Precautions 44. Protocol Pre-approval 45. Protocol Scope 5 5.1. Intended Location Of Use 5 5.2. Strains To Be Validated 5 5.3. Documentation 6 5.4. Comments 66. Verification Of Counts And Purity 7 6.1. Materials 7 6.2. Method 7 6.2.1. BioBall Preparation 7 6.2.2. Inoculation Of Agar Plates And Incubation (Initial Counts) 8 6.2.3. Verification Of 8hr Stability At 2-8c 9 6.2.4. Interpretation Of Results 10 6.2.5. Acceptance Criteria 10 6.3. Documentation 11 6.4. Comments 11 6.5. Section Review And Approval 117. Confirmation Of Organism Identity 12 7.1. Materials 12 7.2. Method 12 7.2.1. Macroscopic And Microscopic Identification 12 7.2.2. Biochemical / Molecular Identification (Optional) 13 7.2.3. Acceptance Criteria 14 7.3. Documentation 14 7.4. Comments 14 7.5. Section Review And Approval 148. Final Review And Approval 15 8.1. Final Comments 15 8.2. Protocol Review And Approval 159. Discrepancies 15

31. About BioBall MultiShot 550The pharmaceutical industry is required to perform QC with inocula containing less than 100 cfu for several tests including growth promotion, sterility and method validation.

Once validated, BioBall may be used as a precise, reliable and cost effective alternative to traditional reference culture methods such as seed lot maintenance systems.

BioBall contains a precise number of microorganisms in a water soluble ball delivering unprecedented accuracy for Quantitative Microbiological Quality Control. Lot specific certificates of analysis confirming counts and organism identity are supplied with each lot number.

BioBall MultiShot 550 has a batch mean of between 500 and 600 cfu with a standard deviation less than or equal to 10% of the batch mean. When re-hydrated with 1.1mL of re-hydration fluid provides 10 x 100uL doses, each containing 50 cfu.

It has an 8 hour stability when re-hydrated with BioBall Re-Hydration Fluid and stored between 2 to 8C, making it ideal when several controls are needed at the same time or within the same day.

BioBall is available in a wide range of microorganisms, including equivalent strains to those required by the British Pharmacopoeia, (BP), European Pharmacopoeia, (Ph.Eur.), Japanese Pharmacopoeia, (JP) and United States Pharmacopoeia, (USP). BioBall reference strains are recommended by BTF for use as an alternative to traditionally derived reference cultures in the following pharmacopoeial chapters:

British Pharmacopoeia & European Pharmacopoeia

Appendix XVIA Tests for Sterility, (Ph.Eur 2.6.1)

Appendix XVIB Tests for Microbial Contamination, (Ph.Eur. 2.6.12, 2.6.13)

United States Pharmacopoeia

Microbiological Examination of Non-sterile Products: Microbial enumeration tests

Microbiological Examination of Non-sterile Products: Tests for specified Microorganisms

Sterility Tests

Validation of Microbial Recovery from Pharmacopoeial Articles

Microbial Enumeration Tests Nutritional and Dietary Supplements

Microbiological Procedures for Absence of Specified Microorganisms Nutritional and Dietary Supplements

Japanese Pharmacopoeia

35. Microbial Limit Tests

36. Microbial Limit Test for Crude Drugs

54. Sterility Test

All reference strain identifications are confirmed by genetic typing.

BTF as a Reference Materials producer is the first company worldwide accredited under the ISO Guide 34 Standard for quantitative microbiologial reference standards, including BioBall.

BioBall products have up to 24 months* shelf life when stored frozen.

*Depending on the organism.

42. About this protocolThis protocol outlines the process used to demonstrate the suitability of BioBall as a replacement for traditionally derived culture collections. The protocol within is designed to meet the requirements of regulatory bodies, with regards to documentation and traceability of the verification exercise.

The verification process involves five main steps;

1. Pre-approval of the verification protocol by quality representatives and personnel executing the protocol, (section 4).

2. Definition of the scope of the verification, including intended location of use and reference strains to be tested, (section 5).

3. Verification of BioBall organism counts, strain purity and 8hr stability at 2-8C, (section 6).

4. Identification of BioBall organisms, (section 7).

5. Final review and approval of verification protocol, (section 8).

Verification is based on adequate recovery of each reference strain grown on culture media petri dishes. Once growth is evident the recovery is enumerated and purity visually confirmed. The organism recovered can then be further identified by traditional methods.

Once completed this protocol should be kept on file for future reference. A reference identifier may be entered in the box on the cover of this protocol for ease of reference.

3. Safety precautionsBioBall MultiShot 550 contains viable and potentially pathogenic bacteria and should only be handled by experienced laboratory personnel trained in the safe handling of these micro-organisms. The number of micro-organisms contained within BioBall MultiShot 550 is low. However, BioBall MultiShot 550 should be handled as if potentially infectious. Refer to your national safety guidelines. After use, dispose of packaging in accordance with appropriate biohazard disposal practices. Do not use the product if the packaging is damaged. Do not use the product after the expiry date. The product should be used according to the procedure described in this verification protocol. Any modifications may affect the results.

4. Protocol pre-approvalBefore commencing this protocol, personnel responsible for performing the verification should be nominated and approval should be sought from the companies quality department. This ensures that the content of this document meets any validation policies that may be effective in the intended location of use.

Name (Print) Title Signature Date

Protocol to be performed by

Protocol reviewed by

Protocol approved for use by

55. Protocol scopeRecord below the details of the site at which BioBall is to be used.

5.1 Intended location of use

Company Name: __________________________________________________________

Address: __________________________________________________________

__________________________________________________________

__________________________________________________________

__________________________________________________________

__________________________________________________________

Department: __________________________________________________________

SIGN / DATE_____________________________

5.2 Strains to be validated

Indicate in the table below which BioBall strains are to be validated as part of this protocol. Comments may be made in section 5.4 regarding organisms not included in this protocol.

Table 1. Strains included in BioBall Verification Plan.

SIGN / DATE_____________________________

*Strains are sourced from NCTC, NCPF and ACM. ATCC is a trade mark of the American Type Culture Collection.

OrganismBioBall MultiShot 550

Product CodeDesignation* Included (Yes/No)

Aspergillus brasiliensis (niger) 56011 NCPF2275 / ATCC16404

Bacillus subtilis 56012 NCTC10400 / ATCC6633

Candida albicans 56013 NCPF3179 / ATCC10231

Clostridium sporogenes 56014 NCTC12935 / ATCC11437

Escherichia coli 56016 NCTC12923 / ATCC8739

Pseudomonas aeruginosa 56017 NCTC12924 / ATCC9027

Salmonella abony 56018 ACM 5080 / NCTC 6017

Staphylococcus aureus 56019 NCTC10788 / ATCC6538

65.3 Documentation

The documentation as listed in table 2 below should be attached to the end of this protocol. Documentation for BioBall is available from www.bioball.com. Additional documentation may be added if required.

Document Title Document filed (YES / N/A)

BioBall Material Safety Data Sheet

Table 2. Documentation

SIGN / DATE_____________________________

5.4 Comments

SIGN / DATE_____________________________

76. Verification of Counts and Purity

6.1 Materials

Required BioBall strains, (section 5.2)

BioBall Re-Hydration fluid, (Catalogue No. 56021)

Calibrated 100L pipette and sterile tips

Tryptone soy agar plates, (TSA), or equivalent

Sabouraud dextrose agar plates, (SDA), or equivalent

Columbia +5% blood agar plates or equivalent

Vortex

Sterile spreaders

Incubators (20 -25C & 30-35C)

6.2 Method

6.2.1 BioBall preparation

1. Using table 3 below, record the batch number of each strain to be used. A Certificate of Analysis should also be retained from the BTF website www.btfbio.com for each batch and should be attached to the end of this protocol.

Organism Catalogue No. Batch Number Certificate of Analysis

Aspergillus brasiliensis (niger) 56011

Bacillus subtilis 56012

Candida albicans 56013

Clostridium sporogenes 56014

Escherichia coli 56016

Pseudomonas aeruginosa 56017

Salmonella abony 56018

Staphylococcus aureus 56019 Table 3. Organism batch numbers and Certificates of Analysis

SIGN / DATE_____________________________

2. Label the BioBall Re-Hydration Fluid with the spare label included in the box of BioBall MultiShot 550.

3. Remove cap from BioBall Re-Hydration Fluid.

4. Remove the stopper from the glass vial containing the BioBall.

5. Tip the BioBall into the BioBall Re-Hydration Fluid, replace the cap and wait 30 seconds. Note: BioBall Re-Hydration Fluid needs to be at room temperature when the BioBall is added. It is important NOT to pour the rehydration fluid into the glass vial. Each re-hydrated BioBall in 1.1 mL BioBall Re-Hydration Fluid contains 10 doses of 100 L

6. Vortex for 5 seconds.

7. Use in aliquots of 100 L.

8. Discard the final 100 L

9. Can be used up to 8 hours after re-hydration, if the re-hydrated BioBall is stored in a refrigerator at 2 to 8C. Re-vortex the re-hydrated BioBall for 5 seconds before each use.

SIGN / DATE_____________________________

86.2.2 Inoculation of agar plates and incubation (initial counts)

1. Select correct type of agar plate for each reference strain according to table 4 below. Label plates accordingly with the organism name and date of inoculation.

2. Using a 100L pipette, inoculate the surface of 5 agar plates with 100L of rehydrated BioBall solution onto each plate.

3. Using a sterile spreader spread the solution evenly across the surface of the agar.

4. Repeat steps 1 to 3 for all desired organisms.

5. Once completed, transfer all inoculated plates and incubate at the temperature, time and environmental conditions indicated in table 4 below.

6. Record the time and date incubated in table 4 below.

7. Replace the cap onto each strain used and place the remainder of the re-hydrated BioBall solution into a refrigerator at 2 to 8C. Record the time and date at which the solution is refrigerated in table 4 below.

Organism Agar IncubationTime/Date Incubated

Performed by (Initial)

Time/Date removed

from incubator

Performed by (initial)

Time/Date refigerated

Performed by (initial)

Aspergillus brasiliensis (niger) SDA20-25C

5 d

Bacillus subtilis TSA3035C

24 4 hrs

Candida albicans SDA20-25C

5 d

Clostridium sporogenesColumbia

+5% blood

3035C 48 4 hrs (Anaerobic)

Escherichia coli TSA3035C

24 4 hrs

Pseudomonas aeruginosa TSA3035C

24 4 hrs

Salmonella abony TSA3035C

24 4 hrs

Staphylococcus aureus TSA3035C

24 4 hrs

Table 4. Organism strains, agar types and incubation conditions, (initial counts).

96.2.3 Verification of 8hr stability at 2 to 8C

1. Following 8 hours of refrigeration at 2 to 8C remove all suspensions from the refrigerator. Record the time and date at which the solution is removed from the refrigerator in table 5.

2. Vortex each suspension for 5 seconds.

3. Select correct type of agar plate for each reference strain according to table 5 below. Label plates accordingly with the organism name, date of inoculation and 8hr Stability.

4. Using a 100L pipette, inoculate the surface of 5 agar plates with 100L of rehydrated BioBall solution onto each plate.

5. Using a sterile spreader spread the solution evenly across the surface of the agar.

6. Repeat steps 3 to 5 for all desired organisms.

7. Once completed, transfer all inoculated plates and incubate at the temperature, time and environmental conditions indicated in table 5 below.

8. Record the time and date incubated in table 5 below.

Organism Agar Incubation

Time/Date removed

from refrigerator

Performed by (Initial)

Time/Date Incubated

Performed by (Initial)

Time/Date removed

from incubator

Performed by (initial)

Aspergillus brasiliensis (niger)

SDA20-25C

5 d

Bacillus subtilis TSA3035C

24 4 hrs

Candida albicans SDA20-25C

5 d

Clostridium sporogenesColumbia

+5% blood

3035C 48 4 hrs (Anaerobic)

Escherichia coli TSA3035C

24 4 hrs

Pseudomonas aeruginosa TSA3035C

24 4 hrs

Salmonella abony TSA3035C

24 4 hrs

Staphylococcus aureus TSA3035C

24 4 hrs

Table 5. Organism strains, agar types and incubation conditions, (8hr stability).

10

6.2.4 Interpretation of results

1. Remove all plates from the incubators, recording the time and date that the plates were removed in the applicable table above.

2. Inspect each plate for growth, counting all colonies present on each of the five replicate plates. Whilst doing so, taking note of the purity of each plate, and record all results in the relevant table below.

OrganismPlate count (CFU) Mean count

(CFU)

Pure Growth

(YES/NO)

Recorded by Sign/Date1 2 3 4 5

Aspergillus brasiliensis (niger)

Bacillus subtilis

Candida albicans

Clostridium sporogenes

Escherichia coli

Pseudomonas aeruginosa

Salmonella abony

Staphylococcus aureus

Table 6. Results for organism counts and purity, (initial counts)

OrganismPlate count (CFU) Mean

count (CFU)

Pure Growth

(YES/NO)

Recorded by Sign/Date1 2 3 4 5

Aspergillus brasiliensis (niger)

Bacillus subtilis

Candida albicans

Clostridium sporogenes

Escherichia coli

Pseudomonas aeruginosa

Salmonella abony

Staphylococcus aureus

Table 7. Results for organism counts and purity following refrigeration of solution at 2-8C

6.2.5 Acceptance criteria

1. The counts obtained for all organisms must be within 10 -100 colony forming units on each agar plate, (CFU) for the recovery to be valid.

2. Cultures must be visibly pure that is all colonies should be macroscopically identical with no evidence of plate contamination.

11

6.3 Documentation

The documentation as listed in table 8 below should be attached to the end of this protocol. Documentation for BioBall is available from www.btfbio.com. Additional documentation may be added if required.

Document Title Document filed (YES / N/A)

QC certificates for all BioBall strains used in the verification test

QC certificates for SDA media used in the verification test

QC certificates for TSA media used in the verification test

QC certificate for BioBall Re-Hydration Fluid (Catalogue No. 56021)

Table 8. Documentation

SIGN / DATE_____________________________

6.4 Comments

SIGN / DATE_____________________________

6.5 Section Review and Approval

Once the results for counts and purity are recorded a review of the results against the acceptance criteria should be made.

Counts acceptable Yes / No*

Purity acceptable Yes / No*

* circle as applicable

Name (Print) Title Signature Date

Results reviewed by

Results approved by

12

7. Confirmation of Organism Identity

7.1 Materials

Inoculated agar plates from section 6 (with growth)

Glass slides

Gram stain reagents

Microscope

Biochemical microorganism identification kit, e.g. API, Vitek II (optional)

DNA/RNA microorganism identification kit (optional)

7.2 Method

7.2.1 Macroscopic and Microscopic Identification

1. Using plates showing growth from section 6 of this protocol, examine growth present for each organism type, recording both macroscopic, (colony) and microscopic characteristics in table 9 below. Microscopic identity should be performed using staining techniques appropriate to the organism.

Organism Macroscopic descriptionConforms (YES/NO)

Microscopic description

Conforms (YES/NO)

Performed by

Aspergillus brasiliensis (niger)

White / pale yellow mycelium, spherical, filamentous colonies, become black

with conidia (spores)

Long, smooth conidiophore, biseriate phialides, round vesicle.

Bacillus subtilisIrregular, dull, dry, erose, opaque cream

coloniesGram positive rods

Candida albicans Circular, smooth, convex and bright whiteOvoid/circular budding

cells

Clostridium sporogenesGrey/translucent irregular colonies, dry, dull

in appearance, with an undulate marginGram positive rods

Enterococcus faecalisOff-white, entire, circular, smooth, glistening,

convexGram positive cocci

Escherichia coliCircular, low convex, smooth, entire,

translucent and cream in colourGram negative rods

Pseudomonas aeruginosaIrregular, glistening, can have a greenish

colour on large colonies, with amucoid edgeGram negative rods

Salmonella abony Entire, circular, cream, smooth, glistening Gram negative rods

Staphylococcus aureusEntire, smooth, golden yellow, circular

colonies with sometimes also a paler cream/white colony variant

Gram positive cocci

Table 9. Macroscopic and microscopic colony morphology.

13

7.2.2 Biochemical / Molecular Identification (optional)

1. As an option, the laboratory may wish to further identify each microorganism strain isolated. Details of the method used and the results should be entered into the space below.

SIGN / DATE_____________________________

14

7.2.3 Acceptance criteria

1. Identifications of all organisms should match the descriptions listed in table 9.

2. If biochemical or molecular methods are used to identify each strain, the results must correctly confirm the organism identity.

7.3 Documentation

The documentation as listed in table 10 below should be attached to the end of this protocol. Documentation for BioBall is available from www.bioball.com. Additional documentation may be added if required.

Document Title Document filed (YES / N/A)

Biochemical / molecular identification result reports (if applicable)

Table 10. Documentation

SIGN / DATE_____________________________

7.4 Comments

SIGN / DATE_____________________________

7.5 Section Review and Approval

Once the results for culture identity are recorded a review of the results against the acceptance criteria should be made.

Identifications acceptable Yes / No*

* circle as applicable

Name (Print) Title Signature Date

Results reviewed by

Results approved by

15

8. Final review and approval

8.1 Final Comments

SIGN / DATE_____________________________

8.2 Protocol Review and Approval

Once sections 5 to 7 are completed a review of the protocol against the acceptance criteria should be made.

Sections 5 to 7 completed and signed Yes / No*

All supporting documentation attached Yes / No*

Protocol complete Yes / No*

* circle as applicable

Name (Print) Title Signature Date

Protocol performed by

Protocol Reviewed by

Protocol approved by

9. DiscrepanciesAny discrepancies discovered during this protocol should be addressed here. Technical information and advice regarding protocol discrepancies may be obtained from your bioMrieux sales representative.

SIGN / DATE_____________________________

bioMrieux S.A.69280 Marcy lEtoile - FranceTel. 33 (0) 4 78 87 20 00Fax. 33 (0) 4 78 87 20 90www.biomerieux.comwww.biomerieux-industry.com

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