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."
Scope 范围
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". 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
生物制药生产的微生物控制策略
Bioburden for sterile operations
无菌操作的微生物负荷
Evaluation of bioburden excursions in non-sterile biological manufacturing processes
1. Microbial Control System 微生物控制系统
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?"
问题（i）“微生物负荷的标准下限是什么?”小于1的微生物负荷标准不现实吗?（由于测试的环境、耗材和设备）?那么标准下限应该是什么呢?小于3 ?小于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?"
（ii）“关于微生物负荷限度：对在D级、C级和B级生产的原液样品进行无菌取样，在未分级的微生物实验室进行检测。偶尔，取样或操作的培养皿上会出现几个菌落。在这种情况下，将微生物负荷限度设置为<1 CFU/10ml有意义吗？或者有什么推荐？”
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.
回答：逐步递进地设置限度。在接近过程结束时设置更严格的限度，对于冷冻的原液，设置为10cfu /10ml？
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?"
问题（i）“你们是否对每个设定的取样点进行了微生物负荷方法适用性测试？或者你是否采用了基于最差条件样品（中间产品/缓冲干扰检测的可能性）的方法来覆盖其它一些步骤，并基于风险评估减少适用性测试工作?”
(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?"
（ii）“监管机构和检查员——他们对整个缓冲液/原料药/药品生产过程中的微生物负荷控制的监管期望程度是什么，这些都是基于风险吗?”
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.
回答：是的，对每个设定的采样点进行方法适用性测试。采用基于风险的方法设定采样点，考虑到（i）工艺设备的配置，包括微生物负荷过滤器的位置，以避免在检测污染物时可能出现的盲点，或（ii）开放的生产步骤和周围环境，或（iii）用于使假定微生物失活的调节步骤的潜在影响（例如，极端pH值的调整或溶剂/洗涤剂的添加），或（iv）工艺的促生长能力。
Question:"Should buffers which are received sterile filtered be tested for bioburden before being used in manufacturing? We already test for endotoxin."
问题：经除菌过滤的缓冲液在用于生产前应进行微生物负荷检测吗？我们已经检测了内毒素。”
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.
回答：没有任何规范要求这样做。如果缓冲液的无菌性是强制性的，那么这应该通过证书来确认或者通过检测来证实。
Questions: (i) "Should there be alert levels for all bioburden IPCs taken from aseptic production?"
问题：（i）“是否应该对无菌生产中所有的微生物负荷中间过程控制设置警戒水平?”
(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?"
（ii）“对于C-D级洁净区的无菌生产，你是否仍然建议为所有中间过程控制步骤设置警戒水平?”还是只有在A-B级进行无菌生产时才需要对IPC步骤设置警戒水平?”
Answer: Yes.
回答：是的
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?"
问题：“pH值被操纵的工艺（如病毒灭活）能否掩盖其中微生物负荷或内毒素的存在？我的意思是，有没有可能微生物负荷在病毒灭活之前的微生物负荷分析中被检测到，而不是在病毒灭活结束之后?”
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.
回答：是的，必须考虑调节步骤（例如，极端pH值的调整或溶剂/洗涤剂的添加）对假定微生物负荷的潜在失活的潜在影响。
Question:"Do you always sample and test for endotoxins in parallel to Bioburden? If not why?"
问题：“你们总是同时取样并检测内毒素和微生物负荷吗?”如果不是，为什么?”
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.
回答：不总是，但在大多数情况下是的。注：EMA药品、原料药、辅料和直接接触药品容器灭菌指南不要求在除菌过滤前进行内毒素检测，只要求进行微生物负荷控制。
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?"
问题：如果所有IPC步骤都要求设置警戒水平，那么它是否适用于所有产品阶段（临床I-III阶段、PPQ和商用）？如果只生产了少量批次，如何建立I-III阶段和PPQ阶段的警戒水平?”
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.
回答：是的。在开发过程中使用临时限度，直到产生足够的历史数据。或者，对于不经常生产的产品（例如开发中），可以使用类似工艺的数据。
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?"
问题：“由于在过滤除菌前对微生物负荷进行采样，产品还未处于无菌状态，所以一些检查员希望适用于生物制品原液的所有期望同样也适用于滤前样品。这是相关的吗？你有什么建议吗？”
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.
回答：逐步提高限度的理念也应该应用于DP生产。因此，制剂生产所适用的限度不应比原料药所规定的限度宽松。逐步提升限度。
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?"
问题：“那么，微生物负荷（及其副产品）对使用过的生产设备的影响如何呢？例如微生物计数，是否有任何指南/理由来评估树脂/UFDF膜在生产过程中再次使用的安全性?如果结果是TNTC（不可计数），没有计算可以执行，有没有办法（例如某种空白运行研究）来防止这种副产品的影响?”
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.
回答：清洁和消毒操作应该足够严格，以去除污染物和任何副产品。清洗和消毒措施的有效性必须在清洗验证中得到证明，必须在日常工作中建立对清洗和消毒措施的监测。
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?"
问题：“关于生产保持时间的限度，定义每个工艺步骤的保持时间是否重要？或者从微生物学的角度来看，整个保持时间（灭菌开始前的所有过程步骤）是重要的吗？”
Answer: Yes. A maximum hold time for each process step must be defined and validated.
回答：是的。必须定义和验证每个工艺步骤的最大保持时间
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)?"
问题：“你们描述了评估药物成分和原料药生产商污染风险的控制措施。据你所知，是否对生产原料药或用于非无菌药品（干剂型）的原料的生产设施有环境监测要求？”
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.
回答：如果为将要进行的生产定义了一个卫生区域，那么必须满足该卫生区域的要求。
原文来自ECA网站，点击“阅读原文”可以查看原文。
