Bioburden is defined as quantity of bacteria on an un-sterilized surface. It is the measure of contamination on any surface before being sterilized. Since bioburden is a major concern for the safety and efficiency of pharma products, bioburden is frequently monitored to keep it under acceptable limits by employing various prevention techniques.
There is no specific classification of surface that is prone to the dangers of bioburden. Any surface, such as equipment, part of the equipment, liquid, solid, or any other material that comes in contact with pharma products or takes part in the manufacturing process, can be affected by bioburden.
For an effective bioburden control strategy, bioburden monitoring is necessary and critical. It provides the necessary data, actual conditions, and bioburden specifications to investigate the cause and source of bioburden. This data is helpful to researchers in devising contamination control strategies.
Some regulating guidelines regarding bioburden and its monitoring
Regulatory bodies also remain concerned about bioburden, and its control strategy, which they specifically review during their routine inspection.
Some common guidelines related to Pharmaceutical bioburden that provides a helpful resource in understanding and monitoring bioburden include the following.
21 CFR 21.113
The 21 CFR 211.113, is the regulation related to Good Manufacturing Practices – GMP, and refers to finished pharmaceuticals. Its subpart – F, covers the production and process controls and emphasizes the importance of written procedures for controlling bioburden or microorganisms.
It states that,
Written procedures that effectively prevent lethal microorganisms in Pharma products must be established and followed for both sterile and non-sterile.
It also emphasizes the importance of validating the aseptic process and states that.
Additionally, sterile products should also include validation of aseptic and sterilization processes.
21 CFR 211.165 (b)
21 CFR 231.165 is the Good Manufacturing Practice – GMP guideline for finished Pharmaceuticals and is related to the testing and release of pharma products before distribution in the market.
This part mentions that there shall be adequate laboratory testing procedures for each batch of Pharma products to prevent hazardous microorganisms.
21 CFR 211.84 (d)(6)
21 CFR211.84 is the Good Manufacturing Practices for finished pharmaceuticals and deals with the testing and approval of rejection of component's drug product containers and closures.
Party (d) of 21 CFR 211.84 represents guidelines related to the examination and testing of samples. Serial number 6 of this regulation is relevant to the testing of microorganisms or bioburdens and according to this serial number.
Each lot of component, drug product container, or closure that is prone to contamination should be tested for microbial bioburden before use.
USP Chapter 111 Microbiological Examination of nonsterile products: Acceptance Criteria For Pharmaceutical Preparations And Substances For Pharmaceutical Use
USP chapter 1111 provides acceptance criteria for non-sterile pharma products. The acceptance criteria are based on the outcome of a microbial test or an average of repetitive tests, where applicable.
There are two units for acceptance criteria - Total Aerobic Microbial Count (TAMC) and Total Combine Yeast and molds Count (TYMC)
USP Chapter 61 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
This test provides guidance for the quantitative determination of a Bioburden. This test can be used for the determination of mesophilic bacteria and fungi. Additionally, this test cannot be used to determine viable microorganisms.
The USP chapter 61 describes the membrane filtration method or plate count method. According to this chapter, the test method depends upon the product's nature and the microorganisms' limit.
USP Chapter 62: Microbiological Examination Of Nonsterile Products: Tests For Specified Microorganisms
The test described in USP chapter 62 determines whether specific microorganisms exist in a given sample or surface.
USP Chapter 1115: Bioburden Control of Non-Sterile Drug Substances And Products
This chapter covers the requirement of bioburden control strategies for non-sterile drug products that are critical for human consumption. According to this chapter, drug products that are potentially at risk of microbiological contamination include the following
How bioburden monitoring helps in Pharma processing
Bioburden monitoring is an effective tool to detect and determine microbial contamination, which can then be used to implement preventive and form the basis of corrective measures.
Let's discuss some helpful ways bioburden monitoring helps in Pharma processing.
The primary function of bioburden monitoring is to investigate the Pharma process for the presence of any microorganisms. It helps personnel to quantify and qualify hazards that become frequent in Pharma product manufacturing. The investigation can also detect shortcomings or vulnerabilities in the attached services and processes.
Some ways bioburden monitoring helps in the investigation is as under
It helps to determine the type of bioburden present in a sample. Because there can be various categories of microorganisms that can become apparent during different stages of the pharma product manufacturing process. There are different corrective and preventive measures for every type, and accurately categorizing the type of bioburden helps determine accurate corrective and preventive measures.
It also helps determine the quantity of microorganisms in a given sample.
The above two characteristics of bioburden monitoring are critical in accurate product investigation and remedial actions for a safe and effective Pharma product.
Helps in determining preventive measures
Bioburden monitoring helps in devising and shortlisting preventive measures because, there are different preventive measures for any microorganism category and quantity,. Accurately selecting preventive measures also helps to prevent unnecessary and irrelevant measures that are not effective. Thus making the contamination control measures cost-effective.
Accurate estimation also helps during validation studies because accurate bio-burden quantification and qualification are the basis of effective validation studies.
Bioburden identification methods
Let's discuss some common bioburden identification methods employed in the pharmaceutical industry.
Gram Staining Test
Gram stain test determines the type of microbial contamination in a given sample. This test is evaluated under a microscope, providing two types of information - Gram staining and cell morphology. These two types of information form the basis for determining the type of microorganisms.
This test is performed by introducing a colored stain (violet crystal) that turns the cell wall into violet color. Iodine, which sticks to the added violet crystal and fixes the stain. A decolorizer, comprising of Alcohol and acetone, is added, which causes the thin-walled cell to lose the violet stain and is called a Gram-negative microorganism.
The thick-walled cell of the microorganism makes it difficult for the violet crystal to escape The cell wall. As a result, the violet color crystal is retained and is called a Gram-positive microorganism. The structure of the cell is analyzed by microscope.
A red stain is added to the Gram-negative microorganism to counter-stain it at the end of the test, which causes them to appear red.
The gram-negative microorganism is considered risky and is a source of endotoxin.
Spore staining tests are used to identify microorganisms that have spores-like characteristics. Microorganisms havehave spores-like structures, are highly resistantstructures, have no metabolic activity, and are formed due to adverse environmental conditions. They are resistant to heat, dehydration, and various chemical properties, which contribute to the survival of these microorganisms.
In this testing method, a green colored stain is used to stain the spores. The spores are formed inside a vegetative cell and are called Endospores. The cell wall of endospores is thick-walled and resistant to the characteristics mentioned above.
After the sample is stained with a green-colored solution, it is counter-stained with a red-colored solution. Once the green-colored stain penetrates the cell wall, it is rinsed with a red color aqueous solution under the effects of a factor of heat. If spores are present in the sample, its color will not change, indicating the resistance by the spores, thus identifying the presence of spores.
Phenotypic methods identify the type of microorganism in a given sample. They are routinely used in the testing of water, chemical, and Pharma testing laboratories.
This test identifies the microorganism by identifying its gene expression properties, such as size, shape, and color.
This method requires a traditional sample collection process and incubating for the required temperature to provide suitable conditions for microorganism growth. After the passage of a specific time, the samples are analyzed for the presence and identification of microorganisms.
This method comes up with disadvantages related to traditional methods, such as
However, despite above mention limitations, phenotypic methods are still being used in the pharma industry and are proven to detect bioburden.
Mass spectroscopic methods
Mass spectroscopy is an analytical method for the determination of bioburden in a given sample.
The method used is called the Matrix Assisted laser Desorption Ionization time of flight (MALDI-TOF) method. It enables direct detection of microorganisms from the sample, in contrast to other methods that require sample preparation before applying the sample to the test method.
The sample is mixed with a substance called matrix in this test method. The mixture is then applied to the cell of the instrument. After application, the sample is bombarded with a nitrogen laser. The mixture containing the sample absorbs the laser and is vaporized. This makes the sample ionized and gains the electrical charge.
The electrical field inside the instrument guides the ionized mixture into a time of light spectrometer, which separates them according to mass-to-charge ratio. Each ion is identified as a function of a specific microorganism.
The method of bioburden detection by mass spectroscopy requires less time, typically in the order of minutes. As a result, large tests can be performed in less time, yielding high throughput.
Other bioburden monitoring techniques must not be completely abolished when using this technique. Because if the instrument is not available such as during instrument service, it can halt the testing processes. Products purified water is used for cleaning purposes.