Summary of FDA Review for In-Process Bioburden Limits in BLA Approvals (2019–2024):

The FDA has consistently required and reviewed detailed information regarding in-process bioburden limits as part of BLA submissions for biologics. The following key elements are routinely expected and assessed:

1. Definition and Monitoring of In-Process Bioburden Limits

• Pre-established bioburden limits must be set for critical manufacturing steps. These limits are to be justified and provided in the BLA submission, typically in section 3.2.S.2.4 of the Common Technical Document (CTD) ^{2611152026313539}.
• Bioburden sampling is expected to occur prior to any 0.2 µm filtration step, which is often the sterilizing filtration step ^{2611152026313539}.
• Qualified bioburden test methods must be used, and method qualification/validation data should be included ^{211152031353966}.

2. Data Submission Requirements

• Bioburden and endotoxin data from at least three process qualification (PPQ) lots must be submitted to demonstrate process consistency and control ^{2611152026313539}.
• Microbial data from hold time validation studies: Data from three successful product intermediate hold time validation runs at manufacturing scale are required. Bioburden and endotoxin levels before and after the maximum allowed hold time must be monitored and shown to be within limits ^{2611152026313539}.
• Chromatography resin and UF/DF membrane lifetime studies: Protocols and acceptance criteria for bioburden and endotoxin samples during these studies must be provided. Samples should be taken at the end of storage prior to sanitization ^{2611152026313539}.
• Shipping validation studies: Information and summary results regarding bioburden and endotoxin control during shipping should be included ^{2611152026313539}.

3. Release Specifications and Method Qualification

• Drug substance bioburden and endotoxin release specifications must be provided ^{2611152026313539}.
• Summary reports and results from bioburden and endotoxin test method qualification studies for in-process intermediates and the drug substance are required. If compendial methods are used, brief descriptions and references must be included ^{261115202631353966}.

4. Numerical Limits and Acceptance Criteria

• The FDA has cited a pre-sterile filtration bioburden limit of not exceeding 10 CFU/100 mL as an example of an acceptable limit ³⁰⁵¹⁶⁵.
• The actual numerical limits may vary depending on the product and process, but justification and validation are always required.

5. Deficiencies and Post-Marketing Commitments

• Applications have been found deficient if microbial limits for in-process intermediates are not provided or if the method suitability for bioburden testing is inadequate ^{1016194347505466}.
• The FDA has issued post-marketing commitments requiring sponsors to provide additional bioburden method suitability data for in-process samples from additional lots ⁶³⁶⁶.

6. Facility and Process Controls

• The FDA reviews facility readiness, including registration, inspection status, and the adequacy of contamination control strategies ^{2611152026313539465964}.
• Adequate descriptions of facilities, equipment, environmental controls, and cleaning procedures are required to support microbial control claims ⁴⁶⁵⁹⁶⁴.

7. Guidance and Reference Documents

• The FDA refers sponsors to the 1994 Guidance for Industry, "Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products," for further details on aseptic processing and microbial control expectations ³⁰⁵¹⁶⁵.

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Representative Example (2023):

"Bioburden and endotoxin levels at critical manufacturing steps should be monitored using qualified bioburden and endotoxin tests. Bioburden sampling should occur prior to any 0.2 um filtration step. The pre-established bioburden and endotoxin limits should be provided (3.2.S.2.4). Bioburden and endotoxin data obtained during manufacture of three process qualification (PPQ) lots (3.2.S.2.5). Microbial data from three successful product intermediate hold time validation runs at manufacturing scale. Bioburden and endotoxin levels before and after the maximum allowed hold time should be monitored and bioburden and endotoxin limits provided (3.2.S.2.5)." ^{2611152026313539}

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In summary:
The FDA’s review of in-process bioburden limits for BLAs in the last five years has focused on:

• The establishment and justification of numerical bioburden limits at critical process steps,
• The submission of supporting data from process validation and hold time studies,
• The use of validated test methods,
• The demonstration of ongoing process control and facility readiness,
• Addressing deficiencies and, where necessary, requiring post-marketing commitments to ensure robust microbial control throughout the manufacturing process.

References:
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