Humanwell Pharmaceutical Ethiopia PLC

Form No.: SOP/SL/015/F/01

Sterility Test Validation Record Form

Attachment (A)

MICROBIOLOGY ANALYSIS RECORD

STERILITY TEST MEMBRANE FILTRATION METHOD

Product/Bulk/Raw Material Name: Code No. :

Batch/Lot No.: Analytical No.:

Test Method: Validation Reference:

Sampled on: Sampling Remarks:

Analyzed on: Analyzed by:

Inspection of sample units for cracks, defective crimps, and cloudy solution:
________________________

Sample exterior surfaces sprayed with sterile 70% EPA M 3% H2O2-------- for ∫ 10 min
prior to test.
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

Exterior surfaces of steritest and all items relating to the test, sanitized with sterile 70%
EPA M 3% H2O2-----

Type of canister: TTHA LA2 10  TTGS LA2 10  TTHV AB2 10  TTHA PC2 10  TLHV
SL2 10  Lot No.

Sample preparation ________________ / Reconstituted with 10 ml of sterile Using______

No. of tested samples: Tested as: 1 group  3 groups  Individual samples 

Holding time before filtration _________Sampling preparation remarks

Type of rinsing fluid: A  Fluid D  Lot No. Sterility Reference:

Step Pump speed

Pre-wet the membranes with 25 mL of sterile

rinse solution

Product testing

Clear the product from the tubing and

canisters

Rinse the tubing, canisters, and membranes

Additives to the diluent Penicillinase Other neutralizing agent

Add Media to the Canisters

Media used Lot Sterilit Pump Negative control

 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

No. y Ref. speed

Fluid Confo Not

Thioglycolat rm conform
e

TSB Confo Not

rm conform

Under LAF, with sterile scissors, cut the tubing approximately 2

cm above the clamp, fold over and insert the tubing into the top
air vents of the canisters

Accidental events during test:

Negative control using

Negative product control using

Incubation: Incubator Memmert K896-0002 at 32°C Incubator Precision WB 83326658 at 22°C

Result of environmental microbiological monitoring

Monitoring Results Microbial

 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

Positi Positi Positi

on 1 on 2 on 3 Identification

RCS+ of sterility room

Settle plates in LAFH

Surface monitoring of
LAFH

Surface monitoring of
sterility room

Operators’ gloved hand

plates

Operators’ gowns

Results of sterility test

Test 3 days 7 days 14 days 21 days

TSB

Fluid Thioglycolate

Negative control

Product negative
control

*T = turbidity C = clear

Identification of the detected growth:

Stasis test within 3 days
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

TSB Fluid Thioglycolate

Organism Tested with about 100 cfu of each organism

B. subtilis ATCC 6633 Growth No growth Growth No growth

S. aureus ATCC 6538P Growth No growth Growth No growth

C. albicans ATCC Growth No growth Growth No growth

10231

Asp. niger ATCC 16404 Growth No growth Growth No growth

Environmental isolate Growth No growth Growth No growth

Evaluation Conform Invalid test Should be repeated Not conform

Final Comment:

Performed by: Date:

Checked by: Date:

Approved by: Date:

QC Manager

Attachment (B)

MICROBIOLOGY VALIDATION REPORT

STERILITY TEST OF ____________

1. Purpose
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

To validate sterility test method no. _________ membrane filtration for estimation for
sterility test. The recovery of pharmacopoeial and in-house organisms by filtration
method has been tested and to validate that quantity of rinsing fluid is suitable to
neutralize antimicrobial activity of injectable product. The methodology shall not
provide an opportunity for false negative.

2. Scope
Microbial recovery studies of membrane filtration method challenged with less than 100
CFU of each organism listed in USP-25, EP-2002 and inhouse microbial isolates. Two
lots of (Product Name) (Batch Numbers) have been validated in triplicate for each
organism. Validation mimicked the test proper in every detail, such as in the volumes of
media used, quantities and dilutions of product and diluents.
3. References
3.1. USP 25 Sterility test
3.2. USP 25 <1227> Validation of Microbial Recovery from pharmacopoeial articles
3.3. Quality System Regulation, Code of Federal Regulations, 21 CFR 820 (1996)
3.4. Parenteral Drug Association, Technical Report No. 21, Bioburden Recovery
Validation, Vol. 44, No. S3 (PDA, 1990)
4. Conclusion
4.1. (Product Name) can be tested for sterility by membrane filtration with 3 x 300
rinsing fluid A after incubation of 14 days and more than one week.
4.2. The neutralization procedure showed an effective neutralization for the antibiotic
effect (if applicable).

Checked by: Date:

Approved by: Date:

Attachment (B)

MICROBIOLOGY VALIDATION REPORT

 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

STERILITY TEST OF (PRODUCT NAME)

COMPLEMENTARY TEST

Complementary
Tests Procedure Results

Millipore steritest Steritest system Steritest system is valid

validation validation protocol #
TCPU 0 VG 01 Rev. 5

Environmental SOP No.______ All result of active air

control sampling; settle plates;
monitoring RODAC plates, swabs; and
operator’s gloved hand
plates within the acceptance
limit

Sterility of STM No.__________ Lot No. F9AM31473 is

antibiotic sterile (incubated for 21
membrane filter days)

Sterility of STM No.__________ Lot No. R7AM22431 is

Sterisolutest sterile (incubated for 21
days)

Media sterility STM No.__________ Fluid thioglycolate

Millipore Lot No.
H2CN11088 B is sterile
(incubated for 21 days)
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

TSB Millipore Lot No.

H2CN11505B is sterile;
growth appears in all
inoculated media containers
within 2 days

Count of the STM No.__________ The inoculum count of all

challenge challenge organisms in the
inoculum range between 10 and 100
CFU

Media growth STM No.__________ Fluid thioglycolate

promotion test Millipore Lot No.
H1PN04885 A is sterile
(incubated for 21 days)

Sterility test STM No.__________ Sterile, and pass stasis test

after incubation period

Sterility test STM No.__________ Sterile, and pass stasis test

after incubation period

Negative control STM No.__________ Sterile (incubated for 21

(sterile placebo) days), performed by two
analysts (Hamdy & Amal)

Recovery of SOP No.__________ Conform to the acceptance

injured criteria
microorganisms
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

Testing for SOP No.__________ Holding 1h & then rinsing 3

residual times with 300 ml fluid A
antimicrobial nullifies the residual
activity antimicrobial activity of the
product

Validation of SOP No.__________ Two analysts passed the

aseptic working acceptance criteria
technique when
performing
sterility test

Attachment (B)

MICROBIOLOGY VALIDATION REPORT

STERILITY TEST OF (PRODUCT NAME)

TEST FOR BACTERIOSTASIS AND FUNGISTASIS

FINAL REPORT

The filter is pre-wetted with diluent before filtration. 21 vial (Product Name) is reconstituted
with WFI and held for 1 h. In triplicate, the reconstituted sample is filtered through the
membrane filter and washed carefully by 3 x 300mL portions of diluting-fluid A. Inoculate the
final 100-mL portion with less than 100 CFU of each challenge microorganism, and pass through
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

the filter. This filter vessel is then filled with the appreciated medium and incubated for recovery.
A concurrent viable count has been carried out when performing any of these tests using
membrane filtration method and Miles-Misra control method as a check that the working dilution
has been correctly prepared and calculated. After incubation, morphological characteristics of
the detected growth obtained from both methods have been checked.

Acceptance criteria: The test media are satisfactory if visual evidence of growth appears in all
inoculated media containers within 5 days of incubation.

Batch No. ______________________

Initia FTB (32˚C) TSB (22˚C)

Challenge l showed showed
Microorganism count turbidity after turbidity after

Pseud. aeruginosa 65 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 9027

E. coli ATCC 8739 58 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

Bacillus subtilis 76 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 6633

Staphylococcus 85 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h
aureus ATCC 6538

Enterobacter 55 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h
colcacae isolate from
purified water

Micrococcus luteus 58 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

environmentally
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

isolated

Staph. epidermis 49 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

environmentally
isolated

Cl. sporogenes 59 An- 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 11437 Aerobically

Candida albicans 33 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 10231

Aspergillus niger 58 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 16404

All tested organisms showed conform results for (Product Name) using membrane filtration
technique and rinsed with 3 x 300 ml of fluid A.

Attachment (B)

MICROBIOLOGY VALIDATION REPORT

STERILITY TEST OF (PRODUCT NAME)

TEST FOR BACTERIOSTASIS AND FUNGISTASIS

FINAL REPORT

Batch No. ______________________

Challenge Initia FTB (32˚C) showed TSB (22˚C) showed

Microorganism l turbidity after turbidity after
 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

count

Pseud. aeruginosa 65 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 9027

E. coli ATCC 8739 58 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

Bacillus subtilis 76 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 6633

Staphylococcus 85 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h
aureus ATCC 6538

Enterobacter 55 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h
colcacae isolate
from purified water

Micrococcus luteus 58 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

environmentally
isolated

Staph. epidermis 49 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

environmentally
isolated

Cl. sporogenes 59 An- 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 11437 Aerobically

Candida albicans 33 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

ATCC 10231

Aspergillus niger 58 Aerobically 48 h 48 h 48 h 48 h 48 h 48 h

 Humanwell Pharmaceutical Ethiopia PLC
Form No.: SOP/SL/015/F/01
Sterility Test Validation Record Form

ATCC 16404

All tested organisms showed conform results for (Product Name) using membrane filtration
technique and rinsed with 3 x 300 ml of fluid A.