本文围绕微生物控制与检测的策略展开，核心内容可分为以下三大模块，结合 ATCC（美国典型培养物保藏中心）专家观点与行业案例，分析了生物制药领域的微生物管理逻辑：

• 原材料污染
  细胞系：5%-35% 用于生物生产的细胞存在支原体污染；
  检测试剂：DNA 提取试剂盒、PCR 用牛血清白蛋白（BSA）可能藏有细菌或微量 DNA，干扰检测结果；
  非无菌衍生物：鸡蛋、植物淀粉等需排查沙门氏菌、曲霉菌符合 USP <61>/<62 > 标准。

• 生产环境与操作风险
  环境因素：10% 的工艺污染来自洁净室气流如 HVAC 压力异常、过滤器老化；水作为注射剂基础需持续监测，污染物易形成难清除的生物膜；
  人员因素：美国制药行业超百万从业者，人为失误曾导致 80%-90% 的 GMP 偏差，即便自动化改进后仍占 50%；
  交叉污染：取样未遵循无菌技术、生物反应器清洁不彻底，如Sanofi马萨诸塞州工厂因 30 个月内 20% 生物反应器运行污染被 FDA 发警告信。

• 易被忽视的隐患
  低水平 “可存活但不可培养” 微生物：休眠状态下难检测，激活后破坏生产；
  一次性系统（SUS）：供应商未保证无菌如孔洞、组装问题，易被空气传播微生物污染；
  宿主细胞内源性污染：5% 的 CHO 细胞系含内源性病毒基因序列，需验证是否为真实病毒 / 类病毒颗粒；
  工艺添加剂：廉价非无菌缓冲液等可能污染生物反应器。

• 传统检测方法
  生物负荷检测：水溶性产品用 0.45μm 过滤法，但支原体可穿透，需其他方法，高粘度产品（甘油 / 鸡蛋制剂）用平板接种法，需 5 天 - 2 周观察微生物生长，有效但耗时；
  内毒素检测：USP <85> 规定的 LAL 法（鲎变形细胞凝块）、家兔热原试验，多为定性 / 半定量，显色法灵敏度更高但仍非全定量。

• 新兴快速方法
  核心优势：缩短周转时间（如 PCR 检测支原体 4-6 小时出结果，NGS 检测外源病毒 5 天出结果，远快于传统方法的数周），适配细胞疗法（如 CAR-T 短保质期）的快速放行需求；
  
  
• 典型技术：
  rFC 法（重组因子 C）：内毒素激活后释放荧光，实现全定量检测；
  ATP 生物发光法：1 小时内通过 ATP 与荧光素酶反应判断污染；
  流式细胞术 / 激光诱导荧光（LIF）：实时检测、灵敏度达单细胞水平，可区分活 / 死微生物；
  固相细胞计数法：融合过滤与荧光标记，数小时内完成单细胞级检测，适配细胞疗法快速放行；
  巨噬细胞激活检测：通过 ELISA 快速检测内毒素诱导巨噬细胞释放的 IL-6。

• 供应链可靠性：从持 ISO 17034:2016 资质的机构或授权经销商采购，杜绝非正规渠道；
•  储存与传代控制：细菌培养物需低于 - 20℃储存以保活力，优先使用零传代材料，避免传代导致微生物特性改变；
• 标准化与溯源性：对照品需关联 USP/WHO 标准，确保检测结果可追溯，同时选择供应稳定的供应商，避免商业化后更换关键材料引发流程波动。

Microbial contamination represents a significant risk during biomanufacturing. Unlike medicines that can be synthesized chemically using highly controlled materials, biopharmaceuticals require work with dynamic biological systems. Those inputs and their associated growth conditions give biotherapies “unique vulnerability to contamination,” Fang Tian (director of biological content at ATCC) explained to me. Complicating that factor is the ubiquity of microbial life. Ingress at any point in a process “can compromise product quality and efficacy, patient safety, and regulatory compliance,” she continued. “Especially for cell therapies,” which cannot undergo the types of chromatography steps that purify protein drugs, “microbial contamination brings serious risks for patients.” Quality assurance and control (QA/QC) scientists must devise extensive mitigation strategies and process controls to prevent bacterial, fungal, and viral intrusion from a multitude of sources — and from cell-line development through fill-finish.

微生物污染在生物制造过程中是一个重大风险。与那些可以使用高度受控的材料通过化学方法合成的药物不同，生物制药需要借助动态的生物系统开展工作。ATCC（美国典型培养物保藏中心）生物内容主管Fang Tian向我解释道，这些投入物及其相关的生长条件使生物疗法 “对污染具有独特的易感性”。使这一因素变得复杂的是微生物生命的无处不在。过程中任何环节的侵入 “都可能损害产品质量和功效、患者安全以及法规合规性”，她继续说道，“尤其是对于细胞疗法而言”，因为细胞疗法无法进行那些用于纯化蛋白药物的色谱步骤，“微生物污染会给患者带来严重风险”。质量保证和控制（QA/QC）科学家必须制定广泛的缓解策略和过程控制措施，以防止细菌、真菌和病毒从多种来源侵入 —— 并且要涵盖从细胞系开发到灌装完成的整个过程。

Microbiological testing remains a cornerstone of contamination control. However, during our discussion early in 2025, Tian and ATCC colleagues Nilay Chakraborty (principal scientist and head of cytobiology) and Leka Papazisi (principal scientist in product life-cycle management for research and industrial solutions) indicated their sense of a shift in how the biopharmaceutical industry regards such testing. Rather than thinking about microbiology assays as means for reacting to contamination events, quality teams are implementing testing as part of layered, proactive, risk-based approaches that prevent microbes from entering processes and moving through to finished products. Scientists also are leveraging rapid test methods to improve procedures and outcomes.

微生物检测仍然是污染控制的基石。然而，在 2025 年初我们的讨论中，Fang Tian与 ATCC 的同事Nilay Chakraborty（首席科学家兼细胞生物学负责人）和Leka Papazisi（负责研究和工业解决方案的产品生命周期管理的首席科学家）表明，他们认为生物制药行业对这类检测的看法正在发生转变。质量团队不再将微生物检测视为对污染事件做出反应的手段，而是将检测作为分层、主动、基于风险的方法的一部分来实施，以防止微生物进入生产流程并最终进入成品。科学家们也在利用快速检测方法来改进流程和结果。

Below are excerpts from my discussion with Chakraborty, Papazisi, and Tian. After describing potential sources of contamination, they reflect on the role of microbiological testing in a comprehensive and proactive QA/QC framework. They highlight limitations with traditional methods and workflows as well as advantages of emerging rapid methods, especially in the context of cell therapies. Drawing upon work with ATCC, a biological resources center and standards organization, the trio emphasizes the criticality of reliably produced, supplied, and handled reference materials against which to validate test results. And they call attention to opportunities for advancement, including the development of cryopreserved reference materials that can expedite and improve testing.

以下是我与Chakraborty、Papazisi和Fang Tian讨论的节选内容。在描述了潜在的污染源之后，他们思考了微生物检测在全面且主动的 QA/QC 框架中的作用。他们强调了传统方法和工作流程的局限性，以及新兴快速方法的优势，尤其是在细胞疗法的背景下。凭借与 ATCC（一家生物资源中心和标准组织）的合作，这三位科学家强调了可靠生产、供应和处理的参考物质的重要性，这些参考物质可用于验证检测结果。而且他们还提请注意一些改进的机会，包括开发冻存参考物质，这些物质可以加快并改进检测工作。

Reviewing the Risks

回顾风险

What are some possible sources of microbial contamination during bioproduction? What possibilities are overlooked or underestimated?

生物生产过程中微生物污染的一些可能来源是什么？哪些可能性被忽视或低估了？

Papazisi: If we dissect a bioprocess from beginning to end using a risk-based approach, we consider risks from materials inputs and rank them by severity and probability of occurrence. Raw materials are the first consideration if we’re discussing cell-culture processes (1). Studies suggest that anywhere from 5% to 35% of cell lines used for bioproduction have mycoplasma contamination (2, 3). That is a serious problem. Also, when it comes to using nonsterile derivatives from live organisms — e.g., eggs, egg-derived substrates, or starch from plants — we need to check for other pathogens, such as Salmonella, Aspergillus, and others specified in United States Pharmacopeia (USP) <61> or <62>.

Papazisi：如果我们用基于风险的方法从头到尾剖析一个生物工艺过程，会考虑物料投入带来的风险，并按严重性和发生概率对这些风险排序。要是讨论细胞培养过程，原材料是首要考量因素（1）。研究显示，用于生物生产的细胞系中有 5% 到 35% 存在支原体污染（2，3），这是个严重问题。此外，涉及使用活生物体的非无菌衍生物（比如鸡蛋、鸡蛋衍生的底物或植物淀粉）时，我们需要检查是否存在其他病原体，像沙门氏菌（Salmonella）、曲霉菌（Aspergillus）以及《美国药典》（USP）<61> 或 < 62 > 中规定的其他病原体。

A raw-material risk that I was surprised to learn about involves test reagents (4). For instance, materials in DNA-surveillance kits can harbor contaminants. You might have a clean process, and your test method might seem pristine. But if your kit contains bacteria or trace DNA, their presence will skew your conclusions. In personal communications, I also have learned about cases of mycoplasma contamination in bovine serum albumin (BSA) used in polymerase chain reaction (PCR) assays for mycoplasma detection.

一个让我意外的原材料风险与检测试剂有关（4）。比如，DNA 监测试剂盒里的材料可能藏有污染物。你可能有一套清洁的工艺，检测方法看似也很完善，但如果试剂盒含有细菌或微量 DNA，它们的存在会使你的结论出现偏差。在私下交流中，我还了解到，用于支原体检测的聚合酶链反应（PCR）分析里用到的牛血清白蛋白（BSA）存在支原体污染的情况。

Other risks come from manufacturing environments — e.g., airflow, water, and cleanroom surfaces. Ideally, such surfaces would be sterile, although some small level of microbial presence is acceptable. But some studies show that 10% of process contamination comes from airflow in cleanrooms (5, 6). Maybe your heating, ventilation, and air conditioning (HVAC) system does not provide the right pressure, or maybe an air filter is worn out. And because water serves as the basis of many injectable products, continuous water monitoring is critical. Contaminants from all of those sources form biofilms — and microbes that have formed weird structures on your filters and equipment can be difficult to eliminate.

其他风险来自生产环境 —— 比如气流、水和洁净室表面。理想情况下，这些表面应该无菌，尽管存在少量微生物是可接受的。但一些研究表明，10% 的工艺污染来自洁净室的气流（5，6）。可能你的供暖、通风与空调（HVAC）系统没提供合适的压力，或者空气过滤器已经老化。而且，因为水是许多注射用产品的基础，持续监测水至关重要。来自所有这些来源的污染物会形成生物膜 —— 而在过滤器和设备上形成异常结构的微生物可能很难清除。

Employees are a critical source of contamination. In the United States alone, the pharmaceutical industry employs over a million people, who represent many potential causes of good manufacturing practice (GMP) deviations (7). Decades ago, it used to be that 80–90% of deviations derived from human error (7). A 2015 study by writers from the BioPharm Operations Group notes that automation and continual process improvements can decrease that value to about 50% — but definitely not zero (8).

员工是污染的关键来源。仅在美国，制药行业就雇佣了超一百万人，他们是良好生产规范（GMP）偏差的诸多潜在诱因（7）。几十年前，曾有 80% 到 90% 的偏差源于人为失误（7）。生物制药运营小组的作者在 2015 年的一项研究中指出，自动化和持续的工艺改进能将这一数值降低到约 50%—— 但绝对不会降到零（8）。

Meanwhile, cross-contamination can occur when someone performs sampling in two bioreactors and does not follow aseptic techniques or when a bioreactor is not cleaned sufficiently, allowing biofilms to remain at the start of another batch. Those are major issues that still occur frequently and need to be addressed. In 2025, the US Food and Drug Administration (FDA) issued a warning letter to Sanofi site in Framingham, MA, after 20% of bioreactor runs were rejected over 30 months (between January 2022 and July 2024) due to contamination concerns (9).

与此同时，当有人在两个生物反应器中进行取样却未遵循无菌技术时，或者当生物反应器清洁不充分、致使生物膜在另一批生产起始阶段残留时，交叉污染就可能发生。这些是仍频繁出现且需要解决的重大问题。2025 年，美国食品药品监督管理局（FDA）向位于马萨诸塞州弗雷明翰的赛诺菲工厂发出警告信，原因是在 30 个月内（2022 年 1 月至 2024 年 7 月期间），20% 的生物反应器运行因污染问题被拒收（9）。

One final set of risks to consider includes factors from external environments. Pests come to mind: Insects can fly in and out of a facility, bringing possibilities for contamination.

最后一组需要考虑的风险包含来自外部环境的因素。容易想到的是害虫：昆虫能在设施内外飞进飞出，带来污染的可能性。

Regarding overlooked contamination sources, we should mention low-level microorganisms that are viable but not culturable. Such microbes remain dormant until, for whatever reason, they activate and wreak havoc on your manufacturing process. Viable-but-not-culturable microorganisms are the “needle in the haystack.” They are not easy to detect. However, rapid microbiology techniques can help, and with increased sampling and improved sampling plans, you can identify and mitigate such issues quickly.

关于被忽视的污染源，我们应当提及低水平的 “可存活但不可培养” 的微生物。这类微生物会保持休眠状态，直到因某种原因被激活，并对您的生产过程造成严重破坏。“可存活但不可培养” 的微生物就像 “大海捞针”，并不容易检测。不过，快速微生物学技术会有所助益，而且通过增加采样和改进采样计划，您能快速识别并缓解这类问题。

Airborne microbes also can contaminate single-use systems (SUS) for bioprocessing. If a vendor does not ensure sterility — e.g., SUS can have holes and assembly problems — then contamination problems can arise.

空气传播的微生物也会污染生物加工用的一次性系统（SUS）。如果供应商不确保无菌性 —— 比如，一次性系统可能存在孔洞和组装问题 —— 那么污染问题就可能出现。

A growing concern in the field relates to endogenous contamination from host cell lines. An estimated 5% of Chinese hamster ovary (CHO) cell lines contain endogenous viral gene sequences (10, 11). The question is whether such sequences are for true viruses or virus-like particles. Regulatory agencies are becoming increasingly aware of such risks, so the current mandate is for cell lines to undergo rigorous stability testing, especially as users scale up their production processes.

该领域日益受到关注的一个问题与宿主细胞系的内源性污染有关。据估计，5% 的中国仓鼠卵巢（CHO）细胞系含有内源性病毒基因序列（10，11）。问题在于这些序列属于真正的病毒还是类病毒颗粒。监管机构对这类风险的认识日益加深，因此当前的要求是细胞系要进行严格的稳定性测试，尤其是当使用者扩大其生产规模时。

A final overlooked contamination source is process additives. Buying inexpensive material from a vendor that has not ensured its product’s sterility can compromise your bioreactor — and all because you wanted, for instance, just to use a buffer to adjust process pH. Additives are simple yet important considerations.

最后一个被忽视的污染源是工艺添加剂。从没有确保产品无菌性的供应商那里购买廉价材料，可能会危害您的生物反应器 —— 而这全是因为您（比如）只是想用一种缓冲液来调节工艺 pH 值。添加剂是简单却重要的考量因素。

Chakraborty: It is worthwhile to zoom out and highlight why such vulnerabilities matter. The concern relates to a final product’s intended use. Drug developers do not expect microorganisms to be present in their products. Dead or alive, contaminants can change a product’s characteristics.

Chakraborty：把视角放远并强调这类脆弱性为何重要是值得的。这种担忧与最终产品的预期用途有关。药物研发人员不希望其产品中存在微生物。无论污染物是死是活，都能改变产品的特性。

We also should keep in mind that, as the biopharmaceutical industry drives toward automated processes and related standards, the checks and balances in our test procedures will need to change. Nonsterile microbes can get into your new processes, and that will be like riding a train from Washington, DC, to Boston: Once you are on it, you cannot get off. That is another reason for implementing rigorous mitigation.

我们还应当记住，随着生物制药行业向自动化流程及相关标准迈进，我们测试程序中的制衡机制将需要改变。非无菌微生物可能会进入您的新流程，而这就像乘坐从华盛顿特区到波士顿的火车：一旦登上，就无法下车。这是实施严格缓解措施的另一个原因。

Risk-based assessment of microbial risks seems to be a straightforward process, but are there better or worse ways to approach mitigation? How effectively do companies translate microbiology QC testing into risk mitigation?

基于风险的微生物风险评估似乎是一个直接的过程，但在实施缓解措施方面，是否有更好或更差的方法呢？企业将微生物质量控制（QC）测试转化为风险缓解的效果如何？

Chakraborty: Risk mitigation involves certain checks and balances, as I mentioned. Let’s say that downstream processes are set. In a risk-based approach, we might specify that if a certain microorganism is present during QC testing, we will count its colonies and find out what is happening in the process.

Chakraborty：正如我提到的，风险缓解涉及某些制衡机制。假设下游流程已确定。在基于风险的方法中，我们可能会明确规定：如果在 QC 测试中存在某种微生物，我们将对其菌落进行计数，并查明流程中发生了什么。

Two factors will influence that QC testing, one being processing time. We need time to grow microbiological cultures, find out whether microorganisms are present in samples, and determine whether those present are dangerous. But how long does it take to determine that something is wrong in the process? If a QC testing process has “inertia” built into it — e.g., for preparing and growing cultures — then by the time that a problem is discovered, your process might have moved on to subsequent steps — or your bioprocess material may have been waiting for confirmation. Such risks must be accounted for, and opportunities are available to improve QC testing by cutting down processing time.

有两个因素会影响该 QC 测试，其中一个是处理时间。我们需要时间来培养微生物、查明样品中是否存在微生物，并确定存在的微生物是否具有危险性。但要确定流程中出现问题需要多长时间呢？如果 QC 测试流程存在 “惯性”—— 比如，在制备和培养微生物方面 —— 那么当发现问题时，您的流程可能已经进入后续步骤，或者您的生物加工材料可能一直在等待确认。必须考虑到这类风险，而且存在通过缩短处理时间来改进 QC 测试的机会。

The second factor is strain authentication. You need authenticated reference materials to ensure that what you believe to be a dangerous strain is, in fact, dangerous. We can improve QC testing by implementing authenticated microbial cultures.

第二个因素是菌株鉴定。您需要经过鉴定的对照品，以确保您认为具有危险性的菌株实际上确实具有危险性。我们可以通过使用经过鉴定的微生物培养物来改进 QC 测试。

Papazisi: We should note that QC testing implies testing of finished products — that is, quality control. But risk-based approaches against contamination relate inherently to quality assurance. That function includes examination of raw materials, process monitoring, environmental monitoring, training protocols, and many other considerations besides finished-product testing. It would be a disservice to our biopharmaceutical colleagues not to emphasize assurance because so much work goes into confirming that a process will result in products with the required sterility and integrity. We should remember that QC testing is just one part of a bigger strategy.

Papazisi：我们应当注意到，QC 测试意味着对成品的测试 —— 也就是质量控制。但针对污染的基于风险的方法本质上与质量保证相关。该职能包括对原材料的检查、过程监测、环境监测、培训方案，以及除成品测试之外的许多其他考量因素。如果不强调质量保证，会对我们生物制药行业的同事造成不利，因为要确认一个流程能生产出具有所需无菌性和完整性的产品，需要投入大量工作。我们应当记住，QC 测试只是更宏大策略中的一部分。

As Nilay said, testing must be performed with reliable reference materials — e.g., a culture type referenced by the USP. Regulatory agencies strongly recommend that validation be performed with USP microbiological standards, especially for regulatory filings. You would need to validate your process further if you wanted to use another microorganism.

正如尼莱所说，检测必须使用可靠的对照品 —— 例如，《美国药典》（USP）提及的培养物类型。监管机构强烈建议使用 USP 微生物标准开展验证工作，尤其是在提交监管申报资料时。若想使用另一种微生物，就需要进一步验证工艺。

All told, process validation must be robust. You need to ensure that you are truly mitigating contamination risks, no matter who is working on a process and under whatever conditions.

总的来说，工艺验证必须稳健。要确保无论由谁、在何种条件下开展工艺工作，都能切实降低污染风险。

Tian: The biopharmaceutical industry generally agrees that it is smarter to focus on preventive work covering an entire manufacturing process than it is to rely on tick-off checkboxes. Some organizations might think about QA/QC in terms of final-product testing for regulatory requirements rather than addressing risks proactively. But we want to guarantee that products will be safe and sterile — and that, even if regulatory requirements change, our products will be up to the test. Proactive attitudes toward microbiological risk maybe should be emphasized even more in today’s industry than they already are.

田：生物制药行业普遍认为，专注于涵盖整个生产流程的预防性工作，比依赖 “打勾式” 检查更明智。一些机构可能从满足监管要求的成品检测角度考虑质量保证 / 质量控制（QA/QC），而非主动应对风险。但我们希望确保产品安全且无菌 —— 并且即便监管要求改变，我们的产品也能通过检测。在如今的行业中，对微生物风险的主动态度或许应该得到比现在更多的重视。

Methods Matter

方法很关键

What methods are available for microbiological testing?

有哪些微生物检测方法可用？

Papazisi: We can think of microbiology methods in terms of what must be tested and whether the approach is a traditional or rapid method. Let’s focus on traditional assays first — e.g., compendial tests for bioburden. For water-soluble products, that often involves passing samples through 0.45-μm filters.

Papazisi：我们可以从 “必须检测什么” 以及 “方法属于传统方法还是快速方法” 的角度来思考微生物学方法。先关注传统检测方法 —— 例如，药典规定的生物负荷检测。对于水溶性产品，通常要让样品通过 0.45 微米的滤膜。

Mycoplasma species are small enough to pass through such filter pores, making that method inappropriate for their detection. But such filters should trap other typical bacteria that are listed in the USP chapters. Filters are placed in a culture medium and observed for microbial growth over as few as five days and as long as two weeks, which is effective but not ideal.

支原体的体积小到足以通过这类滤膜的孔隙，因此该方法不适用于检测支原体。但这类滤膜应能截留《美国药典》各章节中列出的其他典型细菌。将滤膜置于培养基中，观察微生物生长情况，时间短则 5 天，长则两周，这种方法有效但并不理想。

The choice of test method depends on the product. Filtration is effective for water-based solutions, but formulations based on glycerol or egg are much more viscous, necessitating plating methods. For instance, you can mix solution with growth medium as it cools down, or you can drag material across a coated plate, and you wait to see what grows.

检测方法的选择取决于产品。过滤法对水基溶液有效，但基于甘油或鸡蛋的制剂粘性大得多，因此需要采用平板接种法。例如，可在溶液冷却时将其与培养基混合，或者将材料在涂有培养基的平板上涂抹，然后等待观察有哪些微生物生长。

An interesting problem emerges when companies test finished products that contain preservatives. Such excipients impede microbial growth, so contamination might not manifest during testing. The question, then, is about how to remove the preservatives. One method is serial dilution. Alternatively, you can use compounds that neutralize a product’s preservatives so that you can observe what microorganisms grow. But you need to validate such methods.

企业检测含有防腐剂的成品时，会出现一个有趣的问题。这类辅料会抑制微生物生长，因此污染可能在检测过程中不显现。于是问题就变成了如何去除防腐剂。一种方法是连续稀释。或者，可使用能中和产品防腐剂的化合物，以便观察有哪些微生物生长。但需要对这些方法进行验证。

Traditional methods for endotoxin testing are listed in USP <85> (12). Such approaches are usually qualitative or semiquantitative. One option is the Limulus amebocyte lysate (LAL) assay: In the presence of endotoxins, horseshoe crab amebocytes create a gel clot. An older approach is rabbit pyrogen testing. Chromogenic methods can provide greater sensitivity and reliability than the LAL and rabbit pyrogen tests, but even those approaches are only semiquantitative.

内毒素检测的传统方法列于《美国药典》<85>（12）中。这类方法通常是定性或半定量的。一种选择是鲎变形细胞溶解物（LAL）检测法：存在内毒素时，鲎的变形细胞会形成凝胶凝块。更古老的方法是家兔热原试验。显色法可比 LAL 和家兔热原试验提供更高的灵敏度和可靠性，但即便这些方法也只是半定量的。

Some methods for rapid testing use recombinant factor C (rFC), a synthetic form of the protein responsible for clotting in LAL assays. Because rFC is designed to release a fluorescent product when activated by endotoxins, the presence of endotoxins can be detected much more quantitatively than it can using the LAL approach.

一些快速检测方法使用重组因子 C（rFC），它是 LAL 检测中负责凝血的蛋白质的合成形式。由于 rFC 被设计为在被内毒素激活时释放荧光产物，因此内毒素的存在能比用 LAL 方法更定量地被检测到。

Mycoplasma testing should begin with analysis of master cell banks. Culture processes must start with clean cell lines. Ideally, you can wait, say, 20 days for results considering that such bacteria are notoriously slow-growing, but if you cannot wait that long, PCR methods can provide answers within four to six hours, including time for extraction.

支原体检测应从主细胞库的分析开始。培养过程必须从洁净的细胞系起步。考虑到这类细菌生长极为缓慢，理想情况下可以等 20 天左右出结果，但如果等不了那么久，PCR 方法可在 4 到 6 小时内（包括提取时间）给出结果。

There is a movement in the biopharmaceutical industry to expand detection of adventitious viruses. Resources from the Parenteral Drug Association (PDA) and related organizations now list about 50 pathogens to test for (1), so viral testing is a big deal. Viruses do not grow as bacteria do, so viral testing traditionally has involved cytopathic assays, in which analysts observe samples (e.g., from cell banks, final products, and so on) over time to detect evidence of viruses damaging plated cells. But cytopathic methods take a couple of weeks for results. Using next-generation sequencing (NGS) can provide results in about five days. Alternatively, PCR can be used to detect adventitious viruses through the presence of viral sequences.

生物制药行业正出现扩大外源病毒检测的趋势。来自注射剂药物协会（PDA）及相关组织的资料如今列出了约 50 种需检测的病原体（1），因此病毒检测是一件大事。病毒的生长方式与细菌不同，因此传统的病毒检测涉及细胞病变检测法 —— 分析人员会随时间观察样品（例如来自细胞库、成品等的样品），以检测病毒损害培养细胞的迹象。但细胞病变方法需要几周才能出结果。使用下一代测序（NGS）可在约 5 天内得出结果。或者，可通过病毒序列的存在，用 PCR 检测外源病毒。

The decision of whether to use traditional or rapid methods for the above testing depends on several factors, including how long you can wait for results and what type of material will be analyzed — e.g., a cell line or fetal bovine serum (FBS). If you lean toward traditional approaches, you should consider assay sensitivity: How much do you want to know about your samples, and how sensitive must your method be? Today, the regulatory threshold is high, so I believe that the pharmaceutical industry is moving toward rapid, comprehensive methods.

对于上述检测是用传统方法还是快速方法的决定，取决于几个因素，包括能等待结果的时长，以及将分析何种材料 —— 例如，细胞系或胎牛血清（FBS）。如果倾向于传统方法，就应考虑检测的灵敏度：希望对样品了解到什么程度，方法必须有多灵敏？如今监管门槛很高，因此我认为制药行业正朝着快速、全面的方法发展。

Other considerations include assay costs and development stage. PCR and other modern methods cost much more than traditional assays do. At the same time, you cannot hold a drug-product lot for 28 days, especially if you are working with an autologous cell therapy. And of course, if your product is still in an early development stage, you can use rapid methods that are not fully validated. But the closer that your product gets to commercial launch, the more that you will need to apply compendial methods.

其他考虑因素包括检测成本和开发阶段。PCR 和其他现代方法比传统检测方法成本高得多。同时，不能将药品批次保留 28 天，尤其是在从事自体细胞疗法相关工作时。当然，如果产品仍处于早期开发阶段，可使用未完全验证的快速方法。但产品越接近商业上市，就越需要应用药典方法。

Chakraborty: If you think about comprehensive microbial QA/QC testing frameworks, a few key characteristics emerge. The first point, as I mentioned before, is that authenticated reference material is a must. That will be your yardstick during testing. Otherwise, you will be lost. Fast operation is another consideration. If you are using automated or semiautomated methods, then you need your systems and materials to be in the right formats for a given use case. The cost of operation should be reasonable. And finally, your assay must be robust. Whether you are working with bacteria or viruses, a comprehensive microbiology framework is important.

Chakraborty：如果思考全面的微生物质量保证 / 质量控制（QA/QC）检测框架，会浮现出几个关键特征。第一点，正如我之前提到的，经过鉴定的对照品是必需的。它会成为你检测过程中的标准。否则，你会不知所措。快速操作是另一个需要考虑的因素。如果使用自动化或半自动化方法，那么你需要让系统和材料针对特定用例采用合适的格式。操作成本应当合理。最后，你的检测方法必须稳健。无论你研究的是细菌还是病毒，全面的微生物学框架都很重要。

What can you say about other emerging rapid methods?

对于其他新兴的快速方法，你有什么看法？

Tian: Such methods have received significant attention lately. That comes in part from the fields of therapeutic antibodies and cell and gene therapies. As advanced therapies become more complex and personalized, QA/QC functions need increasingly robust microbiology test methods. A fast turnaround time is more critical than ever, considering that products such as chimeric antigen receptor (CAR) T-cell therapies have short shelf lives. The reality is that traditional testing requiring >14 days will not meet needs for timely release.

田：这类方法近来受到了极大关注。部分原因来自治疗性抗体以及细胞和基因疗法领域。随着先进疗法变得更加复杂和个性化，QA/QC 职能需要越来越稳健的微生物检测方法。快速的周转时间比以往任何时候都更关键，因为像嵌合抗原受体（CAR）T 细胞疗法这样的产品保质期很短。现实情况是，需要超过 14 天的传统检测无法满足及时放行的需求。

Organizations such as ATCC have been at the forefront of developing rapid test methods. About a decade ago, for instance, we were developing and implementing multiplex PCR mycoplasma testing for all-cell-line manufacturing workflows. And we will continue to explore new and more sensitive assays because we recognize the need to stay “ahead of the game.”

像 ATCC 这样的机构一直处于开发快速检测方法的前沿。例如，大约十年前，我们就在为所有细胞系的生产流程开发并实施多重 PCR 支原体检测。而且我们会继续探索新的、更灵敏的检测方法，因为我们认识到需要保持 “领先优势”。

Papazisi: Keep in mind that methods that have been game-changers in the pharmaceutical industry were not developed initially for such products. Often, they were used for clinical microbiology, food and beverage applications, and so on. PCR testing was created for infectious-disease diagnostics and has since become a routine method in biopharmaceutical operations. Sometimes, the technology is already there and just needs to be applied.

Papazisi：要记住，在制药行业中带来变革的方法最初并非是为这类产品开发的。通常，它们被用于临床微生物学、食品和饮料应用等领域。PCR 检测是为传染病诊断而创建的，此后已成为生物制药操作中的常规方法。有时候，技术已经存在，只是需要加以应用。

Emerging methods for rapid microbiology testing are quite impressive (13–16). One relatively simple assay is based on adenosine triphosphate (ATP) bioluminescence. As microbes grow, they produce ATP, which glows in the presence of luciferase and oxygen. Scientists use that principle to make a test that can save you time and tell you in about an hour whether contamination is present in samples.

新兴的快速微生物检测方法相当令人瞩目（13-16）。一种相对简单的检测方法基于三磷酸腺苷（ATP）的生物发光原理。随着微生物生长，它们会产生 ATP，而 ATP 在荧光素酶和氧气存在时会发光。科学家利用这一原理制作检测方法，既能节省时间，又能在大约一小时内告知样品中是否存在污染。

Other new methods are extremely sensitive — able to find the “needle in the haystack.” Beyond PCR and NGS, those include flow cytometry and laser-induced fluorescence (LIF), both of which can report results in real time and can detect even single microorganisms. Flow cytometry also has the advantage of discerning between live and dead microorganisms. Remember that some are viable but not culturable.

其他新方法极其灵敏 —— 能够找到 “大海捞针” 般的目标。除了 PCR 和下一代测序（NGS），还包括流式细胞术和激光诱导荧光（LIF），这两种方法都能实时报告结果，甚至能检测单个微生物。流式细胞术还有一个优势，就是能区分活微生物和死微生物。要记住，有些微生物是可存活但不可培养的。

Solid-phase cytometry, in a sense, marries flow cytometry and filtration techniques. After a filtration step, retained contaminants are labeled with a fluorescent dye. The method is highly sensitive, down to the single-cell level, and it can be performed within a few hours. Considering the need for rapid release testing for cell therapies, solid-phase cytometry could be the way forward.

从某种意义上说，固相细胞计数法融合了流式细胞术与过滤技术。经过过滤步骤后，被截留的污染物会用荧光染料标记。这种方法灵敏度极高，可达到单细胞水平，且能在数小时内完成。考虑到细胞疗法对快速放行检测的需求，固相细胞计数法可能是未来的发展方向。

Developments specific to endotoxin testing include rFC methods and macrophage-activation assays. Macrophages are part of human innate immunity. When exposed to endotoxins, which macrophages have been trained over many millennia to recognize, those immune cells release proinflammatory cytokines, including interleukin 6 (IL-6). You can expose samples to macrophages and use an enzyme-linked immunosorbent assay (ELISA) to detect even miniscule amounts of IL-6 quickly.

内毒素检测的特定进展包括重组因子 C（rFC）方法和巨噬细胞激活检测。巨噬细胞是人类固有免疫的一部分。当暴露于内毒素（巨噬细胞经数千年进化已能识别内毒素）时，这些免疫细胞会释放促炎细胞因子，包括白细胞介素 6（IL-6）。可将样品暴露于巨噬细胞，然后用酶联免疫吸附测定（ELISA）快速检测极微量的 IL-6。

Bearing the (Reference) Standard for Proactive QA/QC

为主动式 QA/QC 秉持（参考）标准

What are some considerations for selecting reference materials?

选择对照品时需考虑哪些因素？

Papazisi: For validation testing, everything comes back to reliability. First, you need a reliable supply chain for bacterial and fungal cultures. You might expect reference materials to be good because of their source, but suppliers sometimes work through distributors. Ensure that you are buying from suppliers directly or from authorized distributors, not from some organization that claims to have the same material. Also perform due diligence. Check whether an organization holds an ISO 17034:2016 credential for certified reference materials. If a distributor does not have needed certifications, then you will have supply-chain problems and concerns for testing reliability.

Papazisi：对于验证检测，一切都要回归可靠性。首先，需要细菌和真菌培养物的可靠供应链。你可能会因来源而认为对照品质量好，但供应商有时会通过经销商运作。要确保从供应商或授权经销商处采购，而非从声称有相同材料的某机构采购。同时要开展尽职调查，检查某机构是否持有经认证对照品的 ISO 17034:2016 资质。若经销商没有所需认证，会面临供应链问题，且会担忧检测可靠性。

Regarding the reference material itself, you must maintain manufacturer specifications — temperature, for example. Bacterial cultures must be stored at temperatures below −20 °C to ensure their viability. Any deviation from that condition will leave some dead bacteria in your reference material, which will skew the number of bacteria that you expect to find in your test.

关于对照品本身，必须遵守制造商的规格要求（比如温度）。细菌培养物必须储存在低于 -20°C 的温度下以确保活力。偏离该条件会使对照品中出现死细菌，进而导致测试中预期的细菌数量出现偏差。

Passage number is an important consideration for bacteria. If you use material straight from a reputable supplier, then it will have undergone zero passages. But if I borrow material from a colleague’s cultures and grow those bacteria to the numbers that I need for my tests, then those bacteria will have undergone additional passages. The penalty for using such material is that your microorganisms’ characteristics might have changed or faded away, skewing your test results.

传代次数是细菌的重要考量因素。若直接使用知名供应商的材料，传代次数为零。但如果从同事的培养物中借用材料并培养到测试所需数量，这些细菌会经历额外传代。使用这类材料的弊端是，微生物特性可能改变或消失，使测试结果出现偏差。

Such factors should be taken seriously because they affect your test fundamentals. Scientists use analytical reference materials to ensure that instruments can be calibrated accordingly and that methods can be validated. If you select the wrong source for reference materials or if you do not maintain them appropriately, then you should not expect your testing validation to be reliable.

这些因素应被重视，因为它们会影响测试的基本要素。科学家使用分析用对照品来确保仪器可相应校准、方法可被验证。若为对照品选了错误来源，或未对其妥善维护，就不应期望测试验证是可靠的。

Tian: It is worth noting that concepts for bacterial and fungal reference cultures mirror those for animal cell lines, so we apply the same principles to ensure their authenticity and genetic stability. Consider CHO and human embryonic kidney 293 (HEK293) cells, which are not used for microbiological QC testing but play essential roles in biomanufacturing and functional testing of biological products. Such cell lines have hundreds of derivatives from a variety of sources, so confirming cell-line authenticity is critical to manufacturing success and regulatory compliance.

田：值得注意的是，细菌和真菌参考培养物的理念与动物细胞系的理念相似，因此我们应用相同原则来确保其真实性和遗传稳定性。以 CHO（中国仓鼠卵巢）细胞和人胚肾 293（HEK293）细胞为例，它们不用于微生物质量控制（QC）测试，但在生物制造和生物制品的功能测试中起着关键作用。这类细胞系有来自各类来源的数百种衍生物，因此确认细胞系的真实性对制造成功和符合监管要求至关重要。

Many of ATCC’s efforts and publications relate to standards development involve cell-line authentication. We advocate for using methods — e.g., short tandem repeat (STR) analysis — to ensure against cell-line misidentification and contamination. The same principles apply for materials used in microbiology testing.

ATCC（美国典型培养物保藏中心）的许多工作和出版物都与涉及细胞系鉴定的标准制定相关。我们倡导使用诸如短串联重复（STR）分析等方法，以防止细胞系误认和污染。同样的原则也适用于微生物测试中使用的材料。

Scientists focus on the material itself when obtaining reference standards for microbiology QA/QC, but one often-overlooked factor is supply “longevity.” You never want to change your critical raw materials and reference standards after product commercialization. Vendor changes to material specifications can create issues for your whole test regime. Thus, vendor credibility, stability, and longevity are critically important aspects of microbiological testing.

科学家在获取微生物 QA/QC 参考标准时会关注材料本身，但一个常被忽视的因素是供应的 “持久性”。产品商业化后，绝不想更换关键原材料和参考标准。供应商对材料规格的变更会给整个测试体系带来问题。因此，供应商的可信度、稳定性和持久性是微生物测试中至关重要的方面。

Papazisi: A helpful way to think about reference materials is to consider endotoxin testing, material for which must be tied to the USP and/or World Health Organization (WHO) standards for microorganism traceability and measurement. Your test material must be related to those standards. Otherwise, your test lacks validity.

Papazisi：思考对照品的一个有用方式是考虑内毒素测试，其内毒素测试用材料必须与《美国药典》（USP）和 / 或世界卫生组织（WHO）关于微生物可追溯性和测量的标准相关联。测试材料必须与这些标准相关，否则测试就缺乏有效性。

When you work with reputable organizations, you start with materials that are authentic. Such organizations take the time to characterize reference materials. ATCC has efforts underway to sequence all microorganisms in our collection, the primary ones being the USP standards. Of course, we have been performing phenotypic characterization for over 100 years. Now, we are using modern characterization approaches. But we are still working with authentic material that is the same across every culture collection around the world.

与知名机构合作时，会从真实的材料开始。这类机构会花时间表征对照品。ATCC 正在努力对我们收藏的所有微生物进行测序，主要是 USP 标准菌株。当然，我们已经进行了 100 多年的表型表征。现在，我们使用现代表征方法，但仍使用真实的材料 —— 这类材料在全球所有培养物保藏中心都是一致的。

Chakraborty: Because you are working with live organisms, your vendor must be extremely trustworthy. You must use the correct strain — one that did not accumulate mutations that make it different from what you intend to study. And I should emphasize again that there are two "legs" to microbial testing: processing time and microorganism authentication. There is a relationship between those two pillars. Analysts might be using authenticated microorganisms, but their processes could allow their bacteria to change.

Chakraborty：因为你是在和活的生物体打交道，所以你的供应商必须极其可靠。你必须使用正确的菌株 —— 一种没有积累突变、不会变得与你打算研究的菌株不同的菌株。而且我要再次强调，微生物检测有两个 “支柱”：处理时间和微生物鉴定。这两个支柱之间存在关联。分析人员可能在使用经过鉴定的微生物，但他们的流程可能会让细菌发生变化。

That is why ATCC offers MicroQuant single-use, cryopreserved reference materials (17). They are designed to contain the right number of bacteria for QC experiments, as prescribed by USP standards. Because the materials are cryopreserved pellets, they have extremely low processing time (<1 minute) and lack the "inertia" of traditional microbiology workflows. The cryopreserved format also ensures that microorganisms will be from passage zero while enabling compatibility with traditional and automated workflows. These reference materials are manufactured according to all relevant ISO standards (e.g., 9001, 17025, and 17034). We launched the portfolio with 12 microorganisms covering at least two USP chapters, and we are adding microorganisms to provide coverage for the majority of USP chapters.

这就是为什么 ATCC 提供 MicroQuant 一次性冻存对照品（17）。它们被设计为包含符合《美国药典》（USP）标准规定的、用于质量控制（QC）实验的适当细菌数量。因为这些材料是冻存颗粒，所以它们的处理时间极短（小于 1 分钟），并且没有传统微生物学工作流程的 “惯性”。冻存格式还能确保微生物来自零代传代，同时与传统和自动化工作流程兼容。这些对照品是按照所有相关的 ISO 标准（例如 9001、17025 和 17034）生产的。我们推出的产品组合包含 12 种微生物，覆盖至少两个 USP 章节，并且我们正在添加微生物以覆盖大多数 USP 章节。

What are the industry’s next steps for microbial QA/QC?

微生物质量保证 / 质量控制（QA/QC）行业的下一步是什么？

Chakraborty: Leka mentioned that some techniques for biologics QA/QC were developed for other applications. That highlights for me the need for process engineers to identify the right assay and framework for a process need. When an assay gets adopted into a new environment, it is modified. So we need to ensure a match between a new method and a particular process and product. We also need to emphasize robustness.

Chakraborty：莱卡提到，一些用于生物制品 QA/QC 的技术是为其他应用开发的。这让我意识到，流程工程师需要为流程需求确定合适的检测方法和框架。当一种检测方法被应用到新环境中时，它会被修改。因此，我们需要确保新方法与特定的流程和产品相匹配。我们还需要强调稳健性。

Tian: Standardization will be a major concern going forward. ATCC is a standards organization. I see that standardization offers numerous benefits and will be a significant component in the biopharmaceutical industry’s overall approach to microbial testing strategy.

田：标准化将是未来的一个主要关注点。ATCC 是一个标准组织。我认为标准化带来诸多益处，并且将成为生物制药行业微生物检测策略整体方法中的重要组成部分。

Standardizing assay protocols and reference materials should encourage data sharing, facilitating comparison of approaches and data across laboratories. That should foster informed decision-making, continuous improvement, and risk reduction. Enhancing microbial testing will require commitment to innovation and collaboration with regulators and other stakeholders.

标准化检测方案和对照品应能促进数据共享，便于不同实验室之间对方法和数据进行比较。这应该能推动明智的决策、持续改进和风险降低。加强微生物检测将需要对创新的投入，以及与监管机构和其他利益相关者的合作。