Setting and Validating Bioburden Limits

Rob Packard of Medical Device Academy provides an example for setting and validating bioburden limits. 

Last week, I was in Europe reviewing product specifications with a potential contract manufacturer for a medical device implant. Due to the raw materials that the contract manufacturer currently is using for a similar product, bioburden levels are higher than we can accept. The company is using an ISO Class 7 cleanroom for assembly and packaging, which is clean enough for these implants, but the molded components used for the assembly are not clean enough.

Validating Bioburden Limits Setting and Validating Bioburden Limits

In fact, the average bioburden is 220 CFU/device (i.e., colony forming units/device) and the maximum observed bioburden exceeded 500 CFU/device. We want to use a lower dose range of gamma radiation to prevent deterioration of bioabsorbable plastics, but a lower dose range requires that the average bioburden never exceed 100 CFU/device.

There are quite a few Clauses in ISO 13485 that are different from ISO 9001. One example is Clause 6.4–Work Environment. Subsection 6.4(b) states, “If work environment conditions can have an adverse effect on product quality, the organization shall establish documented requirements for the work environment conditions and documented procedures or work instructions to monitor and control these work environment conditions.” This is the applicable clause of ISO 13485 related to setting bioburden limits. Unfortunately, this vague requirement does not explain how to establish or validate bioburden limits.

Rules of Thumb

One of my microbiologist friends recommends using the following “rule of thumb”: +2 sigma for alert limits, and +3 sigma for action limits. This rule of thumb assumes that you are performing data analysis of bioburden, and you have calculated a “sigma” value for the standard deviation. There are a few problems with the “rule of thumb” approach.

First, this method assumes a normal distribution and a controlled process–which bioburden seldom is. Second, the cleanliness you need for your product and the cleanliness your controlled environment is capable of are not always appropriately matched. In my example, we need the finished device to have a bioburden of <100 CFU/device prior to gamma sterilization. Molded parts are essentially bioburden free due to the hot temperatures of the parts ejected from the mold. Unfortunately, molded components attract dust like a magnet. Therefore, how you handle and store molded parts is important to the bioburden of the molded parts.

What Affects Bioburden?

For this example, we have three aspects that are critical to the final bioburden limit of the finished medical devices.

  1. How are the molded parts handled and stored?
  2. Are molded parts cleaned prior to assembly?
  3. What is the cleanliness of the work environment where the device is assembled?

The cleanliness of the molding environment matters, but parts can fall into a container that keeps the parts clean. It also matters how molding machine operators handle the parts. Gloves should be used, and typically the container the parts are in will be placed in an outer bag for storage. It is possible to clean molded parts with ultrasonic cleaning prior to assembly, but if the parts are kept clean after molding this is unnecessary.

For your assembly operation, you need an environment with suitable cleanliness. Sometimes a controlled environment is sufficient. Other times a certified cleanroom is more appropriate. In either case, it is important to control the bioburden in the assembly area to a level that meets the needs of the most critical product assembled in that area. Cleanroom procedures, design of the cleanroom and your cleaning/sterilization processes should match the needs of the product. Fortunately, cleanroom procedures and bioburden limits for cleanrooms are well established in ISO 14644-1 (e.g., for an ISO Class 7 cleanroom, particles ≥ 0.5 microns must be fewer than 352,000). If you have devices of different types in the same manufacturing area, then you need to plan according to the most critical needs.

Validating Bioburden Limits

After you have established your bioburden limits, you need to validate these limits. Once again, cleanroom validation has established ISO Standards to follow. The more challenging validation is validation of the bioburden of component parts and the final assembly. It’s important to validate the component levels first in order to reduce variability of inputs to the final assembly process. Typically the first step is to perform data analysis of other molded parts produced in the same molding area by the same operators. If this data meets your needs for cleanliness, then further measures for controlling bioburden may not be needed. However, if you need to reduce bioburden you might consider measuring parts at critical control points. The goal is to identify where the bioburden is being introduced. This analysis is typical of the type of root cause investigation that is performed when bioburden increases for unexplained reasons as well.

Once the sources of bioburden are identified and quantified, then process controls should be implemented to reduce bioburden. Gloves, double-bagging of product and keeping containers covered during the molding operation are typical risk controls that may be implemented. In order to validate the effectiveness of these measures, you should write a bioburden validation protocol that evaluates each of the following aspects:

  1. lot variability of component bioburden
  2. operator variability for assembly
  3. variability in the cleanliness of the assembly area
  4. number of operators in the assembly area
  5. duration of the manufacturing lots

After you have validated the bioburden limits for the component parts, then the same process should be conducted for the final assembly of product. Sampling of bioburden after transfer to the assembly area, and before assembly begins, should be done. This is important, because often improper storage of components and/or failure to remove and clean outer packaging will contaminate the component parts and your assembly area.

process validation webinar Setting and Validating Bioburden LimitsIf you are interested in learning more about process validation in general, please visit my website to download a new webinar on the topic: CLICK HERE

I will also be publishing a blog in the near future on the topic of documenting sterilization validation for 510k submissions.

Posted in: Validation

Leave a Comment (7) ↓


  1. Jihee March 23, 2015

    Can you elaborate what you mean by you need the products to have less than 100cfu/device to use a lower dosage of gamma irradiation?

    • Rob Packard March 24, 2015

      The limit of 100 CFU/device was an arbitrary limit set for the purpose of discussion. However, there are actual limits for various radiation dose limits. The limit for VDmax 25 kGy is ~1,000 CFU/device, while there is a lower limit for validating at 15 kGy (i.e., VDmax 15). If a company has validated for a different dose of radiation based on “Method 1” in the Standard, then the limit for bioburden will depend upon the dose. All these limits are published in a table found in the ISO Standard for gamma sterilization validation.

  2. Garvey January 8, 2016

    Hi Rob,
    Do you have any suggestions for setting Action levels for low bioburden (100CFU), and SAL 10^-6 (12 log reduction cycle). The trouble I’m having is setting a statistically derived action level that won’t end up being a nuisance. Is it plausible to set a action level based on the number of CFU (~200CFU) that can be accurately counted by the recovery method and knowing the sterilization cycle capability?

    • Rob Packard January 9, 2016

      This is a problem created by “rule of thumb” advice–which I have perpetuated as well. The rule of thumb is: alert limit = +/- 2 sigma and action limit = +/- 3 sigma from the mean. I have received this rule of thumb advice numerous times from experienced auditors–including a microbiologist that is also an accredited person (FDA inspector qualified with a Notified Body). It’s good general advice, but you need some basic assumptions stated about the applicability of the rule of thumb.

      First, you need a normal distribution of the data. Low bioburden levels (i.e., < 100 CFU) are anything but normal. We also don't need a lower limit. We only need to establish upper limits. When you have lots of contamination in a production environment the distribution of bioburden appears normal and very high. When you have a Class 7 clean room with a few people working in the room intermittently, and you gather data quarterly, you do not have normal data. In this case you need to use a scientific rationale that is basic upon your ability to recover the bioburden. Typically your results are "no growth". Depending upon your recovery %, your report might say < 2 CFU/sample. So if you see any growth you hit your alert limit. If you see 10 CFU/sample, you hit your action limit. The reason has nothing to do with statistics, you just want at least a one log cushion to make sure that you are proactively addressing contamination before you hit 100 CFU/sample. I've also seen people perform statistics on the log of the bioburden instead of the actual number. I've seen variations that compromise between the two. I've seen some arbitrary scales. My advice is to keep it simple and explain to the auditors that the data is not normal so you couldn't apply statistical methodology.

  3. Garvey January 11, 2016

    Thank you for taking the time to review and address my question. It’s good to know that there is room to use scientific rational when dealing with a situation like this.

  4. Lyn August 8, 2016

    Dear Rob,

    Can I ask usually we calculate bioburden prior to sterilisation test, do we test for TAMC only or we have to test both TAMS and TYMC? and add both result together to get Total bioburden count?

    Can you elaborate “+2 sigma for alert limits, and +3 sigma for action limits. ”
    For example my biourden limit is 1000cfu/sample, how to set Alert & Action limit?


    • Rob Packard September 28, 2016

      You are correct that adding the bacterial count to the yeast and mold count to obtain a total bioburden count. It is also important to measure both aerobic and anaerobic counts.
      In order to calculate the alert and action limits you perform calculate the mean and the standard deviation of the trend for the bioburden total. The alert limit is the mean plus twice the standard deviation, while the action limit is the mean plus three times the standard deviation.


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