MICROBIO LAB - EXP 17: Microbial Examination of Nonsterile Products and Antimicrobial Preservative Efficacy Testing

Microbial Contamination

Has been a perpetual issue in the manufacturing industry and drug development in the context of microbial attributes

Sterile

• High-risk to end users

• High-level of sterilization and sanitation manufacturing procedures

• Absolute absence of viable microorganisms and their by-products

• Products may be aseptically manufactured or terminally sterilized

Non-Sterile

• Low to moderate risk to end user

• Moderate to low contamination control

• Continuously implementing Current Good Manufacturing Practice (CGMP)

Sterile Products

Such products may be aseptically manufactured or terminally sterilized

Aseptically Manufactured Products

• Products that require extremely high contamination control

• Involve high risk to end user

  • All the processes done in manufacturing a product is sterile from beginning to end

  • Manufactured in cleanrooms (rooms with different compartments that minimize the contamination — consists of Anti-room and Sterile room)

Terminally Sterilized Products

• Products that require high contamination control

• Involve a high-moderate risk to end user

  • Product is only made sterile in the last step

  • Autoclaving

Non-Sterile Products

The presence of certain microorganisms in _____ may:

• Reduce or Inactivate the therapeutic activity of the product

• Alter the physiochemical and organoleptic properties of drugs

• Cause infections and affect the health of the patient

Bioburden

• The number and type of viable microorganisms existing in non-sterile pharmaceutical products

• Absence of objectionable organisms

• Manufacturers should ensure a low _____ of the finished product by continuously implementing Current Good Manufacturing Practices (CGMP) during the manufacture, storage, and distribution of these products.

• Bioburden

• Presence of Gram-Positive Bacteria

• Presence of Gram-Negative Bacteria

Risk Assessment is Based on the:

Presence of Gram-Positive Bacteria

Often implicate human intervention as a major reason for product contamination

• Ex. These bacteria present as normal microflora of the skin

Presence of Gram-Negative Bacteria

• Suggests lack of process control in pharmaceutical environments, particularly those involved in water systems and raw materials

• Gram-Negative Rods

• Most common contaminants of non-sterile pharmaceuticals, regardless of geographical location or time

Microbial Limits Testing

A quantitative enumeration of viable microorganisms and the determination of the absence of specific microorganisms in finished pharmaceutical products and raw materials

Microbial Examination of Nonsterile Products: Microbial Enumeration Tests

Title of USP General Chapter <61>

Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms

Title of USP General Chapter <62>

Microbial Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use

Title of USP General Chapter <1111>

Total Aerobic Microbial Count (TAMC)

Considered to be equal to the number of cfu found using Soybean-Casein Digest Agar

Total Combined Yeasts and Molds Count (TYMC)

Considered to be equal to the number of cfu found using Sabouraud Dextrose Agar

Methods for Microbial Enumeration

• Membrane Filtration

• Plate-Count Methods

  • Pour Plate Method

  • Surface Spread Method

• Most Probable Number (MPN) Method

Membrane Filtration

• Microbiological technique used for the concentration and enumeration of microorganisms in a liquid sample

• used to accommodate large volumes of samples

• Used of the Most Probable Number (MPN) of microorganisms is low

Pour Plate Method

• Not useful for aerobic microorganisms especially of it is non-motile

• Pour-plate method recommended of the bacteria is motile and facultative anaerobe

Surface Spread Method

• Serial Dilution

Most Probable Number(MPN) Method

• Least accurate method for microbial counts

• Reserved for the enumeration of Total Aerobic Microbial Count (TAMC) in situations where no other method is available

• In Product Groups with Very Low Bioburden, it may be the Most Appropriate method.

Test Strain

• Bacillus subtilis (endospore producing)

• Staphylococcus aureus (gram (+))

• Pseudomonas aeruginosa (gram (-))

Growth Promotion of the Media

To test the suitability of the medium to permit the growth of specified microorganisms in the started culture conditions

Neutralization of Antimicrobial Activity

• If your product showed an antimicrobial property, you need to neutralize the antimicrobial property before testing the product

• If no suitable neutralizing method can be found, it can be assumed that the failure to isolate the inoculated organism is attributable to the microbial activity of the product

Negative Control

• To verify testing conditions, a _____ is performed using the chosen diluent in place of the test preparation

• There must be NO GROWTH of microorganisms

  • No Growth = No Contamination

• Ensures that the growth to be seen during the testing of the product comes only from the product itself

Testing of Product

This is performed to validate the dilution and neutralization of antimicrobial property of the product

Growth Promotion Test

Ensures that test microorganisms (which represent the typical bacteria expected to be in contaminated pharmaceutical product) grow in the chosen medium

Suitability of the Counting Method

Performed in the presence of the product to determine the suitability or the “appropriateness” of the product’s diluent, working dilution, additives, neutralization/removal of antimicrobial activity, and counting method

Antimicrobial Preservatives

Substances added to nonsterile dosage forms to protect them from microbial growth or from microorganisms that are introduced inadvertently during or subsequent to the manufacturing process

Antimicrobial Effectiveness Test

• Also known as Preservative Efficacy Test (PET)

• A compendial procedure used to evaluate the performance of those preservatives

• Helps assure that the preservative system is robust enough to prevent the growth of objectionable microorganisms