Bioburden - Question and Answers on Biopharmaceutical Manufacturing

Bioburden control now plays a role in various areas of pharmaceutical and biopharmaceutical manufacturing and quality control. On the one hand, the chapter <1115> "Bioburden Control of Nonsterile Drug Substances and Products" of the USP brings this topic of bioburden into focus for drug manufacturers and focuses on the control of microbial populations throughout the production cycle of excipients, active ingredients and finished drugs.

On the other hand, "bioburden" is also a sterile manufacturing issue. Annex 1 of the European GMP Guide requires: "Bioburden should be monitored prior to sterilisation. Working limits for contamination immediately prior to sterilisation should be established, based on the efficiency of the process to be used. The bioburden test should be performed for each batch, both for aseptically filled products and for terminally sterilised products."

Thirdly, bioburden testing for medical devices manufactured or used in the US is regulated by Title 21 of the Code of Federal Regulations and globally by ISO 11737.


Based on this the ECA Academy had taken up this topic in a special workshop session to look at it from different angles and provide information on the legal background as well as practical examples and strategies for bioburden control. Pharmacopoeia experts, pharmaceutical quality control representatives and testing laboratories compiled key information and highlighted the challenges of bioburden control strategy and how to implement adequate control in companies. From the questions that arose during this workshop, the experts compiled a first Q&A collection. This first Q&A concerns "Biopharmaceutical Manufacturing", compiled by the Chair of the ECA Pharmaceutical Microbiology Working Group, Sven Deutschmann, Roche Diagnostics, and Sebastian Thoelken from Novartis Pharma Stein AG. In this news you will find a first extract from this Q&A document, which covers the following areas:

  • Bioburden testing - guidelines and regulations
  • Microbial control strategy for biopharmaceutical production 
  • Bioburden for sterile operations
  • Evaluation of bioburden excursions in non-sterile biological manufacturing processes

1. Microbial Control System

  • Questions:
    (i) "What is the lower specification for a bioburden? A bioburden with specification less than 1 is not realistic? (due to the environment, consumables and equipment of the test)? So what should be the lower specification? Less than 3? Less than 5?" 
    (ii) "Regarding bioburden limits: Samples from BDS production in Class D, C and B are taken aseptically and analyzed in a non-classified microbiological laboratory. Occasionally, a couple of colonies can appear on plates from sampling or handling. Does it make sense to have a bioburden limit of <1 CFU/10 mL in this setting or what would you recommend?"
    Answer: A stepwise progression of limits is defined. Tighter limits are set closer to the end of the process with ? 10 CFU/10 mL if DS is frozen.

  • Questions:
    (i) "Did you perform a bioburden method suitability test for each defined sampling points? Or did you adopt an approach based on a worst case sample (probability of the intermediate / buffer interferes with the test) to cover some other steps and reduce method suitability test effort based on a risk assessment?"
    (ii) "Authorities and inspectors - to what extend do they expect monitoring the control of bioburden in the complete buffer/drug substance/drug product process, is this all risk based?"
    Answer: Yes, a Method Suitability Test is performed for each defined sampling point. A risk-based approach is used to define the sampling points considering e.g. amongst others (i) configuration of process equipment, including the placement of bioburden reduction filters to avoid possible blind spots in detection of contaminants or (ii) open processing steps and surrounding environment or (iii) the potential impact of conditioning steps (e.g., extreme pH adjustments or solvent/detergent additions) for potential inactivation of  putative bioburden must be considered or (iv) the growth-promoting capability of the process pool.

  • Question: "Should buffers which are received sterile filtered be tested for bioburden before being used in manufacturing? We already test for endotoxin."
    Answer: There are no specifications that require this. If sterility of the buffer is mandatory, then this should also be verified by certificates or demonstrated by testing. 
  • Questions:
    (i) "Should there be alert levels for all bioburden IPCs taken from aseptic production?"
    (ii) "For aseptic production in class C-D clean rooms, do you still recommend alert levels for all IPC steps? Or is alert levels for IPC steps only required when aseptic production in class A-B?"
    Answer: Yes.

  • Question: "Can processes of pH manipulation of the pool (like viral inactivation) mask the presence of bioburden or endotoxins in it? I mean, is possible BB being detected in the BB analysis in a sample of the pool prior to execute the viral inactivation and not after the viral inactivation is ended?"
    Answer: Yes - the potential impact of conditioning steps (e.g., extreme pH adjustments or solvent/detergent additions) for potential inactivation of putative bioburden must be considered.

  • Question: "Do you always sample and test for endotoxins in parallel to Bioburden? If not why?" (note: explicit question to the Novartis colleague)
    Answer: Not always but in most cases. Note: EMA GUIDELINE ON THE STERILISATION OF THE MEDICINAL PRODUCT, ACTIVE SUBSTANCE, EXCIPIENT AND PRIMARY CONTAINER does not request to test for endotoxins prior to sterile filtration. Only a bioburden control is requested.

  • Question: "If alert levels are required for all IPC steps does it then apply to all product phases (Phase I-III, PPQ and commercial)? And how do you establish alert levels for phase I-III and PPQ if only a small amount of batches have been produced?"
    Answer: Yes. Provisional limits are used during development until sufficient historical data has been generated. Alternatively, for products manufactured infrequently (e.g. in development) data from similar processes may be used.

  • Question: "As the Bioburden is sampled before sterilization by filtration the product is not yet sterile so some inspectors want us to apply all expectations that are rather applicable to biological DS. Is this relevant? What would you recommend?"
    Answer: The philosophy of stepwise progression of the limits should be applied for DP manufacturing as well. Hence, the limits applied for Drug Product manufacturing should not be less stringent as the limits defined for Drug Substance. Stepwise progression of limits.

  • Question: "What about the bioburden (and its by-products) impact on used production equipment. In case of microbial counts; there is any guideline/rationale to assess resins/UFDF membranes safety to be used again in the manufacturing process? If the event is TNTC and no calculations can be performed, there is any way (e.g. some kind of blank run study) to defend no impact due to this byproduct?"
    Answer: Cleaning and sanitizing operations should be stringent enough to remove contaminants and any by-products. The effectiveness of the cleaning and sanitizing measures must be proven in a cleaning validation and a monitoring of the cleaning and sanitizing measures must be established in the routine.

  • Question: "Regarding hold time limits for manufacturing, is it important to define hold times for each process step? Or the overall hold time (all process steps until start of sterilization) is what matters from a microbiological point of view?"
    Answer: Yes. A maximum hold time for each process step must be defined and validated
  • Question: "You described controls to assess the risk of contamination for Pharmaceutical ingredients and API manufacturers. To your knowledge, is there a requirement for environmental monitoring of facilities manufacturing API or raw material intended to be used for the manufacturing of non-sterile drug product (dry forms)?"
    Answer: If a hygiene zone is defined for the production in which the production is to take place, then the requirements for this hygiene zone must be fulfilled.

More questions and answers will follow shortly.

If you want to learn more about bioburden, visit the ECA Academy Bioburden Workshop 2022.

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