Cleaning of medical devices is a critical step for evaluating biocompatibility and controlling the microbiological load for sterilization, closely aligning to the ISO 10993-1 requirements. Patient safety is not only related to the specific materials that are being used, but also an understanding of the contaminants or residuals that can be introduced or observed during the manufacturing and handling processes.

The cleaning methods used need to be evaluated to ensure they don’t negatively impact either the biocompatibility or functional performance of the device. Additionally, cleaning validations ensure that the cleaning process removes sufficient levels of contaminants without introducing new contaminants. Cleanliness evaluations of devices can range from testing water purity used in manufacturing processes on a quarterly or monthly basis, confirming that cleaning agent residues are removed, and mitigation of environmental risks. Other areas include risk evaluations of materials used in a cleaning process, identification of potential contaminants throughout the manufacturing process, and ensuring pre-sterilization bioburden levels are sufficiently low.

Device cleanliness evaluations are not limited to manufacturing processes. Reusable medical devices undergo cleaning and disinfection, or sterilization, reprocessing procedures between patients. These reprocessing instructions for use (IFU) must also be validated to prevent any cross-patient contamination in healthcare settings.

Overall, it is the manufacturer's responsibility to provide clean devices to the user. The objective of the cleaning validation is to determine the effectiveness of the cleaning processes to remove physical, chemical, and microbial contaminants or residuals below a defined level. This can be handled with process cleaning steps designed to remove any contamination from the previous step, and a final cleaning before the parts are distributed. ISO 19227 is a guidance often used to evaluate orthopedic implants cleanliness and includes the methods listed below. Additional relevant methods are also outlined.

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microbiological analysis

Bioburden Testing

Prior to final sterilization and packaging, medical devices and pharmaceuticals often must undergo bioburden testing, a quantitative determination of the population of viable micro-organisms either on or in the product. This testing relates to non-sterile medical products as well as pre-sterilization product evaluation. Results from bioburden testing can be utilized to determine proper sterilization levels and procedures.

Microbial Characterization (ISO 11737-1)

Bioburden testing per ISO 11737 may include aerobic bacteria, spores, aerobic fungi, anaerobes, or any combination of the above. Most commonly, the microbiological analysis involves product extraction followed by a membrane filtration, microbial growth on medium, and enumeration of visible colonies. Alternatively, a plate-count method can also be applied (product specific).

Bioburden Recovery Validation (ISO 11135) – Repetitive or Inoculation Method

Bioburden recovery validation is employed to develop and validate a product-specific bioburden testing procedure that will evaluate effectiveness of the procedure, assess antimicrobial properties of the product/sample, and establish a recovery factor for the procedure.

The repetitive (exhaustive) recovery method uses the naturally occurring bioburden of the product to determine the efficiency of the recovery on a test article.Ìý

The spore inoculation method determines the efficiency of recovering the naturally occurring bioburden on a test article by creating an artificial bioburden.

microbiological analysis

Bacterial Endotoxin Testing (BET)

Bacterial Endotoxin Testing (BET) is performed as part of lot release for medical devices and injectable pharmaceutical products. Testing for endotoxins helps ensure that medical devices and implants are safe for human use. It is performed as part of routine monitoring in water systems and incoming materials to ensure that the processes implemented do not contribute to contamination.

Kinetic Chromogenic & Turbidimetric Methods (USP 85)

This method is an in-vitro quantitative assay which photometrically detects endotoxins from gram-negative bacteria, using Limulus Amebocyte Lysate (LAL).This microbiological analysis is applicable to medical devices, sterile and nonpyrogenic assemblies, human and animal parenteral drugs, and other biological products as an important predictor of pyrogenic response.

Validation must be performed with each device and change to process/material. ²ÝÁñÉçÇø’s Kinetic Chromogenic detection limit is 0.005 EU/ml, while the Kinetic Turbidimetric detection limit is 0.010 EU/ml.

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microbiological analysis

Cytotoxicity Testing

Cytotoxicity testing evaluates the toxicity of medical devices and materials. Testing for cytotoxins is required for all medical devices prior to regulatory approval. The testing process is a rapid and highly standardized method that identifies any significant amounts of potentially harmful substances in biomedical materials or devices. The results are useful for screening materials because they serve as a first step to providing evidence of biocompatibility.

Cytotoxicity (ISO 10993-5, USP 87)

ISO 10993-5 and USP 87 are in-vitro qualitative assays used to determine the presence of toxins in medical devices, their components, or raw materials in relation to mammalian cellular viability.

Testing is performed on the final product, representative samples from the final product, or materials processed in the same manner as the final product (see ISO 10993-1). Specimens are prepared in accordance with ISO 10993-12.

Any device or implant that is sterilized before use should be sterilized per manufacturer recommendations prior to testing. If non-sterile test samples are used, the lab will also require that they be checked for bacterial contamination in order to avoid a false assessment of cytotoxicity.

Testing is performed in triplicate with L929 cells.

Methods Commonly Performed

The most performed method of cytotoxicity is MEM Elution method. MEM Elution mimics actual conditions in which medical devices would be used and exaggerates them. Other test methods performed include Agarose Overlay Method and Direct Contact Method. 

Microbiological Analysis

Total Organic Carbon (TOC) & Conductivity

Total Organic Carbon (TOC) testing supports medical device manufacturers monitoring their water for adherence to USP and EP standards. It is also for validating the cleanliness of single and reusable medical devices. It is performed to ensure water systems maintain safe levels of organic compounds and evaluate whether contaminants or residuals from cleaning agents or manufacturing fluids are present.

Microbiological Analysis

Gravimetric Residue Analysis per ASTM F2459

This test method covers the qualitative assessment of the amount of residue obtained from medical device metallic components when extracted by aqueous or organic solvents. Gravimetry on its own may not be sensitive enough for cleanliness assessment, and is often used with supplemental analytical techniques such as FTIR and GC-MS.

International Standards Organization
  • ISO 10993-5
  • ISO 10993-12
  • ISO 11731-1:2018
  • ISO 11135
  • ISO 19227
United States Pharmacopeia 
  • USP <31>
  • USP <61>
  • USP <62>
  • USP <85>
  • USP <87>
  • USP <161>
  • USP <643>
  • USP <645>
  • USP <1231>
Other Standards
  • ANSI/AAMI ST72
  • SM 5310C
  • ASTM F2459-18
  • ASTM F2847-17
  • ASTM F3127-16
  • ASTM F3208-20

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