Protocol Reference: Verification Protocol

Protocol Reference: Verification Protocol

Contents 1. About BioBall Multishot 550 3 2. About This Protocol 4 3. Safety Precautions 4 4. Protocol Pre-approval 4 5. Protocol Scope 5 5.1. Intended Location Of Use 5 5.2. Strains To Be Validated 5 5.3. Documentation 6 5.4. Comments 6 6. 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 2-8 c 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 11 7. 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 14 8. Final Review And Approval 15 8.1. Final Comments 15 8.2. Protocol Review And Approval 15 9. Discrepancies 15 2

1. About BioBall MultiShot 550 The 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 8ºC, 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 <61> Microbiological Examination of Non-sterile Products: Microbial enumeration tests <62> Microbiological Examination of Non-sterile Products: Tests for specified Microorganisms <71> Sterility Tests <1227> Validation of Microbial Recovery from Pharmacopoeial Articles <2021> Microbial Enumeration Tests Nutritional and Dietary Supplements <2022> 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. 3

2. About this protocol This 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 2-8 C, (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 precautions BioBall 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-approval Before 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 4

5. Protocol scope Record below the details of the site at which BioBall is to be used. 5.1 Intended location of use Company Name: Address: Department: 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. Organism BioBall MultiShot 550 Product Code Designation* 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 Table 1. Strains included in BioBall Verification Plan. *Strains are sourced from NCTC, NCPF and ACM. ATCC is a trade mark of the American Type Culture Collection. 5

5.3 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 5.4 Comments 6

6. Verification of Counts and Purity 6.1 Materials Required BioBall strains, (section 5.2) BioBall Re-Hydration fluid, (Catalogue No. 56021) Calibrated 100µL pipette and sterile tips Tryptone soy agar plates, (), or equivalent Sabouraud dextrose agar plates, (SDA), or equivalent Columbia +5% blood agar plates or equivalent Vortex Sterile spreaders Incubators (20-25 C & 30-35 C) 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 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 μl 6. Vortex for 5 seconds. 7. Use in aliquots of 100 μl. 8. Discard the final 100 μl 9. Can be used up to 8 hours after re-hydration, if the re-hydrated BioBall is stored in a refrigerator at 2 to 8ºC. Re-vortex the re-hydrated BioBall for 5 seconds before each use. 7

6.2.2 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 100µL pipette, inoculate the surface of 5 agar plates with 100µL 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 8 C. Record the time and date at which the solution is refrigerated in table 4 below. Organism Agar Incubation Time/Date Incubated Performed by (Initial) Time/Date removed from incubator Performed by (initial) Time/Date refigerated Performed by (initial) Aspergillus brasiliensis (niger) SDA 20-25 C 5 d Bacillus subtilis Candida albicans SDA 20-25 C 5 d Clostridium sporogenes Columbia +5% blood 48 ± 4 hrs (Anaerobic) Escherichia coli Pseudomonas aeruginosa Salmonella abony Staphylococcus aureus Table 4. Organism strains, agar types and incubation conditions, (initial counts). 8

6.2.3 Verification of 8hr stability at 2 to 8 C 1. Following 8 hours of refrigeration at 2 to 8 C 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 100µL pipette, inoculate the surface of 5 agar plates with 100µL 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) SDA 20-25 C 5 d Bacillus subtilis Candida albicans SDA 20-25 C 5 d Clostridium sporogenes Columbia +5% blood 48 ± 4 hrs (Anaerobic) Escherichia coli Pseudomonas aeruginosa Salmonella abony Staphylococcus aureus Table 5. Organism strains, agar types and incubation conditions, (8hr stability). 9

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. Organism Plate count (CFU) 1 2 3 4 5 Mean count (CFU) Pure Growth (YES/NO) Recorded by Sign/Date 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) Organism Plate count (CFU) 1 2 3 4 5 Mean count (CFU) Pure Growth (YES/NO) Recorded by Sign/Date 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 2-8 C 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. 10

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 media used in the verification test QC certificate for BioBall Re-Hydration Fluid (Catalogue No. 56021) Table 8. Documentation 6.4 Comments 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 11

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 description Conforms (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 subtilis Irregular, dull, dry, erose, opaque cream colonies Gram positive rods Candida albicans Circular, smooth, convex and bright white Ovoid/circular budding cells Clostridium sporogenes Grey/translucent irregular colonies, dry, dull in appearance, with an undulate margin Gram positive rods Enterococcus faecalis Off-white, entire, circular, smooth, glistening, convex Gram positive cocci Escherichia coli Circular, low convex, smooth, entire, translucent and cream in colour Gram negative rods Pseudomonas aeruginosa Irregular, glistening, can have a greenish colour on large colonies, with amucoid edge Gram negative rods Salmonella abony Entire, circular, cream, smooth, glistening Gram negative rods Staphylococcus aureus Entire, smooth, golden yellow, circular colonies with sometimes also a paler cream/ white colony variant Gram positive cocci Table 9. Macroscopic and microscopic colony morphology. 12

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. 13

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 7.4 Comments 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 14

8. Final review and approval 8.1 Final Comments 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. Discrepancies Any discrepancies discovered during this protocol should be addressed here. Technical information and advice regarding protocol discrepancies may be obtained from your biomérieux sales representative. 15

06-10 / BTF Issue 3 / This Protocol is made available by biomérieux to the customer as a guide and for information purposes only. The customer understands that they hold responsibility for developing and executing the appropriate protocol, whether they choose to use this information or not, in order to validate the use of BioBall in compliance with the applicable requirements of the regulatory bodies. ATCC is a trademark belonging to the American Type Culture Collection Corporation. BioBall does not contain organisms sourced from The American Type Culture Collection. BioMérieux SA or any of its subsidiaries shall not be liable for any direct or indirect consequence of the use of this information. biomérieux, the blue logo and BioBall are used, pending and/or registered trademarks belonging to biomérieux SA or one of it s subsidiaries/biomérieux SA RCS Lyon 673 620 399 BTF ABN 22 086 488 084. biomérieux S.A. 69280 Marcy l Etoile - France Tel. 33 (0) 4 78 87 20 00 Fax. 33 (0) 4 78 87 20 90 www.biomerieux.com www.biomerieux-industry.com