Microbial testing of Non-Sterile Products: A Brief Overview of Pharmaceutical Microbiology Basics and History, Microbial Control Strategy, Risk Management for Non-Sterile Standards, and Testing and Acceptance Criteria for Non-Sterile Products.
When examining the types of products that carry higher risks, sterile products require a complete absence of microorganisms. This is because they are typically administered into the bloodstream or used within the body, such as medical devices or injections. These products are manufactured under extremely stringent controls due to their high-risk nature.
In contrast, non-sterile products generally pose a moderate to low risk. For these products, a risk assessment is conducted to establish acceptance criteria. Typically, a bioburden limit is set to determine the maximum allowable number of microorganisms that can be recovered from the product. Additionally, the absence of objectionable organisms is a mandatory requirement set by the FDA.
The oral tablets and powdered-filled capsules are the least risky because there is generally little to no water content. If a human takes medicine for a headache, there is a very low chance of any risk. However, on the other hand, if an immune-compromised patient takes an oral tablet, the risk may be slightly higher. Therefore, requirements for non-sterile product testing must be fulfilled.
A pathogen is a microorganism that causes disease in the host. There are two types of pathogens:
Obligate pathogens: Obligate pathogens are pathogens that require a host to recur. They can survive outside the host but cannot proliferate and reproduce. The disease is caused by transmission from one host to another. Examples of these are STDs and tuberculosis.
Opportunistic pathogens: These are pathogens found everywhere—on our skin and in our normal environment. They do not cause problems unless there is a change in that environment, for example, yeast infections.
There was a realization that pharmaceutical products, based on how they were administered, caused problems. So here, we have high drops in all functional dosage forms, and previously, there was much regulation but little control.
Eyedrops are generally multi-dose products, so they have been used multiple times, and there was no limit or volume limit. However, later on, their volume was fixed by implementing a new regulation. There must be a broader spectrum of preservatives for multi-use products that target specific organisms, and in this case, make the product pretty much sterile every time it is used.
Therefore, the requirement of methods to ensure quality products and microbial control during manufacturing is crucial, so monitoring is important. This includes looking at all products having raw materials tested, finished products tested, monitoring the environment, using preservatives for multi-dose products, aseptic manufacturing, sterilization for the right areas, and packaging to ensure that the container closure system is appropriate for the product, in accordance with the regulations.
Non-Sterile Regulation:
21 CFR 211.113 (Control of Microbiological Contamination)
As per the regulation, a written procedure must be established and followed to prevent harmful organisms in drug products not required to be sterile.
A microbial control strategy and risk management are used to identify and prevent microbial contamination that could compromise the quality of products.
Non-Sterile Manufacturing Guidance:
USP 1115 – A risk-based approach to bioburden control of non-sterile products. Here, it is important to identify the sources of contamination in the control of organisms:
- Pathogens, which are part of the normal microbes for humans, could be one of the largest contamination issues during manufacturing.
- Container closures
- People
- Raw materials and ingredients
It is very important to address controlled contamination, as seen with oil. Products containing organic materials pose a much higher risk compared to those using synthetic materials through controlled sterilization. It may be appropriate to treat water or organic materials before they are incorporated into the product, even for non-sterile products, depending on the material. Generally, with non-sterile products, toxins are not as significant an issue.
Testing and Release for Distribution:
“21 CFR 211.84 states that each batch of the drug product container or closure with the risk of microbial contamination must undergo microbiological testing before use.”
“21 CFR 211.165 states that there shall be appropriate laboratory testing, as necessary, for each batch of drugs required to be free from harmful microorganisms.”
USP Testing Method:
USP <61> Test and Purpose:
This test determines the overall bioburden number of CFU (colony-forming units) that can be recovered during the test, specifically targeting bacteria and mold yeast.
The media used for testing are buffer solution and soybean casein digest broth.
Total Aerobic Microbial Count (TAMC): Soybean Casein Digest Agar
Total Yeast and Mold Count (TYMC): Sabouraud Dextrose Agar
The requirements for sample size are generally 10 grams/milliliters, or 1% for smaller batches.
The Sampling Plan should include the following:
It must follow 21 CFR 211.84.
Specify what and where to sample: beginning, middle, and end, or top, mid, and bottom.
Generally, the methods for conducting testing should accurately represent the entire batch in the sampling procedure. They should also specify the required sample size. In some cases, you may need to take 100 milligrams to ensure a representative sample. The procedure should outline the sampling process, including the frequency of testing, and provide specific methods and procedures, including training to prevent contamination.
Additionally, it should detail whether equipment needs sterilization during cleaning and define the requirements for sample labeling, handling, and storage. If the products need to be stored at lower temperatures or experience temperature fluctuations during shipping, these conditions should also be clearly defined in the preparation process.
The beginning sample is prepared in a sterile broth, and the goal is to achieve a homogenous mixture to ensure that a representative amount of samples is tested. For water-sterile products, water-based media is used, but for fatty and gelatinous products, they may contain cell materials.
There are some methods to get those to dissolve or, for example, with transdermal patches, more extraction is performed. There are three methods in the USP Chapter 61. The first is filtration, where the sample preparation is placed. A vacuum is applied through a filter that catches the organisms, and then the filter is placed on the appropriate agar or plates for further analysis.
During the testing of suitabilities, the sample is inoculated with a set number of organisms and types of organisms. These are then run through the test, and growth has to be recovered compared to a positive control, with a recovery rate of 50 to 200 percent. This shows that the testing is valid and that there are no false negatives. However, with USP testing, as with any test, there are limitations. Not all organisms present in the product will grow during this testing.
Related Topic: RABS System
They may be viable but not culturable. General products, non-sterile products, are likely to contain organisms that are very stressed. Placing them in a high-nutrient media may not be sufficient to revive them during suitability testing. A very low number of organisms are inoculated, which helps demonstrate that a small number of organisms can be recovered.
Microorganisms in samples are not uniformly distributed, so when taking samples, it is important to ensure a representative lot is collected. Organisms may only be at the bottom, floating on top, forming a biofilm on the side of a vat, or present as fungal balls floating in the middle. Therefore, it is crucial to obtain a representative sample for accurate testing. Next, we recover the organisms.
USP <62> testing is designed to detect specific organisms. The purpose of this test is to identify the presence of very specific organisms that may be in products. This is a qualitative test, meaning we are only looking for the presence or absence of growth of those specific organisms. The testing is performed using the same preparation described in USP <61>, where the sample is placed in broths after inoculation or directly onto plates. For example, in a Staphylococcus aureus, the sample is to be placed onto Mannitol Salt Agar, which is selective for gram-positive organisms. However, it can be challenging because if Staphylococcus aureus grows, it forms yellow colonies with halos.
In contrast, Staphylococcus epidermidis, another gram-positive organism, does not exhibit the same characteristics. Pseudomonas species, for instance, typically form green colonies and have a grape-like odor. E. coli, on the other hand, does not smell like grapes but forms purple colonies for coliforms due to their ability to metabolize nutrients in the agar, whereas non-coliforms do not produce the same red color. For Salmonella, growth may vary in characteristics, as the screen is for Salmonella species, and the growth may display different traits.
Other examples include the test for Candida albicans, clostridia, and bile-tolerant gram-negative organisms. Once the test is run and characteristic growth is observed, confirmation tests are performed. For example, genetic identification is used to confirm that the organisms recovered are indeed the target organisms. Returning to growth promotion, it is very important for selective and differential broths and agars that growth promotion is performed. This ensures that the media does not allow incorrect organisms to grow and correctly displays the differential characteristics for the target organisms.
A limitation of this test is that it screens only for specific organisms. For instance, if you run a test for E. coli and observe growth, but it turns out not to be E. coli, the organism should be identified in the second step. This is because if the test is capable of detecting the organism, it is possible that E. coli could also grow, indicating a potential issue in the system. However, if the acceptance criteria include organisms not outlined in the USP, a separate screening process must be established.
Next, we will discuss antimicrobial effectiveness testing. As the name suggests, this test evaluates the effectiveness of antimicrobial properties in samples to ensure that preservatives are functioning as intended. The testing must be performed on sterile, non-sterile products that contain antimicrobial preservatives, even if the drug itself is naturally antimicrobial. For sterile products, such as those administered via injection, preservatives inhibit microbial growth during the withdrawal of individual doses. For example, when a vial with a rubber stopper is punctured repeatedly, the preservatives ensure sterility each time.
For non-sterile products, preservatives help control microbial growth during research, manufacturing, and distribution. The preservatives in the sample should be at a level that is effective but not toxic to the user. While preservatives are inherently toxic to microorganisms, their concentration must be carefully controlled to avoid harming the person using the product.
In the USP, antimicrobial effectiveness testing is divided into four categories based on the route of administration and associated risk.
Category 1 includes sterile products, such as injectables and sterile nasal or eye drops.
Category 2 covers non-sterile topical products and those applied to mucous membranes.
Category 3 includes oral products.
Category 4 encompasses products with the lowest risk. During testing, the inoculum is prepared, and the organisms are grown according to USP requirements.
The same method must be followed every time to ensure consistency during testing. Since you cannot use the exact same organism cells, reproducibility requires that the organisms be prepared in the same way each time. The samples are inoculated with the organisms, and a portion is plated at time zero.
The preparation is then incubated and plated at different time points. For example, Category 1 requirements specify not less than a 1-log reduction from the initial calculated counts on day 7, at least a 3-log reduction on day 14, and no increase in organisms from day 14 to day 28.
This ensures the preservatives are effective. Different requirements apply to lower-risk products. It’s important to note that preservatives are not a substitute for good manufacturing practices. You cannot produce samples in inadequate conditions, such as a garage, and simply add preservatives to ensure cleanliness.
Good Manufacturing Practices (GMP) must be followed. The testing focuses solely on the formulation and packaging, so understanding the container closure properties of the sample is essential. As mentioned earlier, microbial variability is a factor since the same cells cannot be used every time. Therefore, it is crucial to have a microbiologist who knows how to perform the testing to ensure consistency and accuracy. Currently, there is no suitability test included in the standard, but guidance is provided in USP 1227.
USP 1111 is a guidance chapter that outlines acceptance criteria for non-sterile products. It is based on the route of administration. For example, for cutaneous products (those used on the skin), the recommendation is less than 200 CFU (colony-forming units). If you are testing for specific organisms, the criteria are stricter: less than 20 CFU for molds and yeast, and the absence of Staphylococcus aureus and Pseudomonas aeruginosa. These organisms are commonly associated with infections if the opportunity arises, making them opportunistic pathogens. However, these are minimum requirements, and additional risk-based assessments should be conducted.
A risk-based assessment is essential to determine appropriate acceptance criteria. This assessment should be performed by a microbiologist familiar with the product, who can identify key risk areas during manufacturing and product use. In some cases, a medical expert may also be needed to evaluate potential patient risks.
When conducting the assessment, the first step is to review critical control points, including the route of administration, the physical characteristics of the product, and the intended user. For example, the site of application (as discussed in USP 1111) helps determine the risk of infection. Products applied to the skin, nasal passages, or transdermal patches generally pose a lower risk compared to rectal or vaginal products.
Naresh Bhakar is the Founder and Author at Pharmaguddu.com, bringing his extensive expertise in the field of pharmaceuticals to readers worldwide. He has experience in Pharma manufacturing and has worked with top Pharmaceuticals. He has rich knowledge and provides valuable insights and data through his articles and content on Pharmaguddu.com. For further inquiries or collaborations, please don’t hesitate to reach out via email at [email protected].