Sterility
| Article | Article No. |
|---|---|
| Sterility Test with membrane filtration (MCE Membrane) | 7130015 |
| Sterility Test with membrane filtration (PDF Membrane) | 7130016 |
| Sterility Test with direct inoculation | 7130005 |
| Sterility Test - Validation per test germ (6 germs are necessary) | 7130020 |
This test is carried out on aqueous, sterile solutions (parenterals, extraction or rinsing solutions and other sterile solutions), water-soluble solids, oily liquids and ointments and creams. A sterile test is performed to show that the products examined are free of microbiological contamination.
Since a sterile test is a destructive test, only samples of a sterilization batch can be examined.
Samples, volume and quantity are specified in the respective pharmacopoeia (e.g. Ph. Eur.: Table 2.6.1-2: Quantities of the product to be examined in the test for sterility and Table 2.6.1-3: Minimum number of items recommended to be tested)
The validity of a sterile test with regard to the sterility of the entire sterilization batch is therefore limited. It is all the greater the more additional parameters are adhered to or checked, such as:
- sterilization is carried out according to a validated procedure
- an adequate amount of bioindicators, for which a reduction of 10⁶ germs can be demonstrated, is reasonable distributed in each sterilization batch
- there is an efficient sampling procedure
PIC/S: Guide to good manufacturing practice for medicinal products Annex 1
USP: United States Pharmacopeia <71> Sterility Tests (current valid version)
Ph. Eur.: European Pharmacopeia 2.6.1. Sterility (current valid version)
EudraLex:
- The Rules Governing Medicinal Products in the European Union Volume 4
- EU Guidelines to Good Manufacturing Practice
- Medicinal Products for Human and Veterinary Use
- Annex 1 Manufacture of Sterile Medicinal Products
Examination of non-sterile products
| Article | Article No. |
|---|---|
| Determination of bacterial load | 7140005 |
| Determination of surface colony count | 7150025 |
| Testing for efficacy of antimicrobial preservation | 7130018 |
| Spore strips test | 7100011 |
This test is carried out to determine the bioburden (determination of population of viable microorganisms) on or in products (especially medical devices), components, raw ingredients or packaging. In particular, the basic working techniques of plating and membrane filtration are described in the internal test specifications and meet the requirements of the pharmacopoeias.
The term "bioburden" refers to the total number of viable microorganisms on a material or product. Since it is not possible to determine the exact bioburden, the determination of bacterial load is carried out according to a defined procedure, which is adapted and validated for the respective product.
Knowledge of bioburden is important with regard to:
- routine monitoring of the manufacturing process for a sterile product
- the determination of sterilization conditions
- the evaluation of the effectiveness of cleaning processes
- testing for efficacy of antimicrobial preservation (s. Ph. Eur. 5.1.3.)
- monitoring the quality of raw ingredients and finished products (s. Ph. Eur. 5.1.4.)
- process validation
Examination of non-sterile products is carried out according to following procedures
DIN EN ISO 11737-1 10-2021: Sterilization of health care products - Microbiological methods - Part 1: Determination of a population of microorganisms on products
Ph. Eur. 2.6.12: Microbiological examination of non-sterile products: microbial enumartion tests
Ph. Eur. 5.1.4: Microbiological quality of non-sterile pharmaceutical preparations
USP 61: Microbial limit tests
Further tests are stability tests regarding antimicrobial preservation according to following procedures
Ph. Eur. 5.1.3: Efficacy of antimicrobial preservation
USP 51: Antimicrobial Effectiveness Testing
In general, the decision tree (DIN EN ISO 11737-1 A.6.1.1 Annexes A and B) is used to select the appropriate procedure.
There are various methods for carrying out each of these 4 steps, which are described in the DIN EN ISO 11737-1 regulation. The methods used by ACILA AG are easy to reproduce and have been determined on the basis of experience. Of the methods described therein, ACILA AG uses:
- Step 1: an elution method or shaking/mixing in the case of direct inoculation depending on the product (see Appendix 1)
- Step 2: By default, 2 methods are used:
- Membrane filtration: with subsequent transfer of the filters to suitable culture media, which enables the growth of aerobic and anaerobic bacteria, as well as yeasts and molds.
- Spread plate method: the direct plating of the product, of an already processed product or of an elution on suitable nutrient media that enable the growth of aerobic and anaerobic bacteria, as well as yeasts and molds.
- Step 3: is carried out using the product inoculation procedure
- Step 4: counting of colony forming units (CFU)
Various methods can also be used to validate the procedures, which are all described in DIN EN ISO 11737-1. ACILA AG carries out the validation using the method of elution of microorganisms, with which a known number of bacteria must be dissolved and recovered from the product after product contamination.
There are no permissible limit values for the initial bacterial count of medical devices. They are to be defined and documented in the course of routine determination, on the basis of the data generated. In general, a bioburden limit < 100 CFU/device is assumed.
Bacterial Endotoxins
The following methods are carried out by ACILA:
1. Ph. Eur.: 2.6.14 LAL Test – Detection or determination of endotoxins of gram-negative bacteria using amoebocyte lysate from horseshoe crab according to:
- 1.1 Gel-clot method
- 1.2 Turbidimetric kinetic method
- 1.3 Chromogenic kinetic tethod
Normative references:
European Pharmacopoeia (Ph. Eur.): 2.6.14 Bacterial Endotoxins
United States Pharmacopeia (USP):
- <85> Bacterial Endotoxins
- <1225> (Validation of Compendial Procedures)
- <1226> (Verification of Compendial Procedures)
- <161> Transfusion and infusion assemblies and similar medical devices.
American National Standard: ANSI/AAMI ST72:2019 (Bacterial endotoxins- test methods, routine monitoring and alternatives to batch testing)
1.1 Gel-clot method:
| Article | Article No. |
|---|---|
| Validation of Bacterial Endotoxin Test, method A, Ph. Eur. & USP (Gel-Clot Test) | 7111017 |
| Bacterial Endotoxin Test (quantitative), method B, Ph. Eur. & USP (Gel-Clot Test) | 7112005 |
| Endotoxin Limit Test, method A, Ph. Eur. & USP (Gel-Clot Test) | 7113004 |
| Endotoxin product characterization, method B, Ph. Eur. & USP (Gel-Clot Test) | 7112024 |
(The article numbers 7112005 & 7113004 are intended for testing on products whose test methods have previously been successfully validated on three production batches.)
In the case of gel-clot methods, a distinction is made between limit value testing (method A) and quantitative determination (method B). These methods are based on a reaction of the amoebocyte lysate at approx. 37 °C with bacterial endotoxins, which leads to gel formation.
1.2 Turbidimetric kinetic method:
| Article | Article No. |
|---|---|
| Validation of Bacterial Endotoxin Test, method C, Ph. Eur. & USP (turbid. kinet.) | 7111018 |
| Bacterial Endotoxin Test (quantitative), method C, Ph. Eur. & USP (turbid. kinet.) | 7112010 |
| Endotoxin Limit Test, method C, Ph. Eur. & USP (kinet.-turbid.) | 7113005 |
| Endotoxin product characterization, method C, Ph. Eur. & USP (turbid. kinet.) | 7112025 |
(The article numbers 7112010 & 7113005 are intended for testing on products whose test methods have previously been successfully validated on three production batches.)
The test is based on a reaction of the amoebocyte lysate at approx. 37 °C with bacterial endotoxins, which is accompanied by turbidity. Turbidity is measured photometrically. The measure of the endotoxin content of a sample is the onset time, i.e. the time that elapses until a desired threshold value is reached. The logarithms for base 10 of the reference standard used and the measured reaction times are plotted against each other. A balancing line is drawn through the points. The endotoxin content of an unknown sample is calculated using this standard straight line. The range of the standard straight line generally includes endotoxin concentrations from 1 to 0.001 I.U./mL. Measurements in this range can be evaluated. This means that the lower limit of endotoxin detection in a test solution is 0.001 I.U./mL.
1.3 Chromogenic kinetic method:
| Article | Article No. |
|---|---|
| Validation of Bacterial Endotoxin Test, method D, Ph. Eur. & USP (chromog. kinet.) | 7111016 |
| Bacterial Endotoxin Test (quantitative), method D, Ph. Eur. & USP (chromog. kinet.) | 7112015 |
| Endotoxin Limit Test, method D, Ph. Eur. & USP (chromog. kinet.) | 7113006 |
| Endotoxin product characterization, method D, Ph. Eur. & USP (chromog. kinet.) | 7112026 |
Endotoxins catalyze the activation of a proenzyme in the Limulus Amebocyte Lysate (LAL). Activation is determined by the concentration of endotoxins. The activated enzyme catalyzes the cleavage of p-nitroaniline (pNA) from the colorless substrate (Ac-Ile-Glu-Ala-Arg-pNA). The concentration of endotoxin in a sample during the reaction time is calculated compared to the reaction time of solutions with known amounts of standard endotoxin (standard curve). The range of the standard curve generally includes endotoxin concentrations from 0.001 to 1.0 I.U./mL.
2. Ph. Eur.: 2.6.32 Test for bacterial endotoxin using recombinant factor C (rFC):
| Article | Article No. |
|---|---|
| Validation of Bacterial Endotoxin Test, rFC method, Ph. Eur. | 7213001 |
| Bacterial Endotoxin Test (quantitative), rFC method, Ph. Eur. | 7213006 |
| Endotoxin Limit Test, rFC method, Ph. Eur. | 7213005 |
| Endotoxin product characterization, rFC method, Ph. Eur. | 7213015 |
This test is based on the gene sequence of the horseshoe crab. A fluorimetric method is used for this purpose. In the field of the determination of bacterial endotoxins, the rFC method has been implemented in the European Pharmacopoeia as a separate chapter (Ph. Eur. 2.6.32).
It offers the following advantages over the LAL test:
- no false-positive results due to the presence of β-glucans
- higher sensitivity
- resource-saving and sustainable alternative method
The measurement range generally includes endotoxin concentrations from 0.001 to 1.0 I.U./mL.
Examination of Water
ACILA carries out purity tests on water (WFI) within the accredited area.
The following methods are accredited:
1. Tests according to European Pharmacopeia (Ph. Eur.):
- Sterility
- Examination of non-sterile products
- Bacterial Endotoxins
- Particle Test
2. Tests according to standard procedures
DIN ISO 11737-1 10-2021: Sterilization of health care products - Microbiological methods – Part 1: Determination of a population of microorganisms on products
DIN EN ISO 9308-1 2017-09: Water quality - Enumeration of Escherichia coli and coliform bacteria – Part 1: Membrane filtration method for waters with low bacterial background flora
DIN EN ISO 7899-2 2000-11: Water quality - Detection and enumeration of intestinal enterococci – Part 2: Membrane filtration method
DIN EN ISO 16266 2008-05: Water quality - Detection and enumeration of Pseudomonas aeruginosa - Method by membrane filtration
DIN EN ISO 6222 1999-07: Water quality - Enumeration of culturable micro-organisms — Colony count by inoculation in a nutrient agar culture medium
DIN EN ISO 11731 2019-03: Water quality - Enumeration of Legionella