1.0 Introduction介绍
The term “low endotoxin recovery” (LER) describes the failure to detect spiked endotoxin in some finished sterile biological drug products when tested using the internationally harmonized compendial Limulus amebocyte lysate (LAL) assay to detect bacterial endotoxin (1–6).Regulators and developers became concerned that compendial methods required by international regulations were unable to confirm the absence of endotoxin in certain drug products, especially troubling considering the inherent risks posed by accidental endotoxin contamination.
低内毒素回收是指对已经添加细菌内毒素标准品的生物制药产品采用国际协调的药典规方法使用鲎试剂进行细菌内毒素检查时加标内毒素检出失败的现象（1-6）。监管机构及开发人员担心国际法规要求的药典方法不能确认特定药物中是否含有内毒素，特别是在考虑到内毒素污染所带来的固有风险。
The failure to recover spiked endotoxins from finished drug products suggested that endotoxin contamination from the manufacturing processes may not be detected at release, thus, pyrogenic products could be distributed for commercial use in patients. This task force, however, searched peer-reviewed, scientific literature, public data on recalls and adverse events, and available copies of the Centers for Disease Control Morbidity and Mortality Weekly Reports and found no instances where a bacterial endotoxin test (BET) failed to detect pyrogenic levels of endotoxin in samples of products released to the market. Some investigational or marketed products were found to be pyrogenic, but those cases were due to non-endotoxin pyrogens or an improperly performed BET (7). Hence, published historical data provide no direct evidence of LAL failing to detect pyrogenic products.
因加入细菌内毒素标准品的药品内毒素回收失败，生产过程中被内毒素污染的含热原产品会因内毒素不能被检出而被放行，含热原的产品因用于商业用途而被患者使用。研究小组对同行评议、科学杂志、召回及不良事件相关的公共数据、疾病控制中心的《发病率和死亡率周刊报道》等进行研究并未发现因细菌内毒素检查（B）失败而导致含热原的上市产品被放行的案例。存在一些被调查发现或上市后发现热原的产品，但是这些案例主要是因为这些热原是非细菌内毒素热原或者细菌内毒素检查方法未能正确执行(7)。已发表的历史数据没有直接证据表明使用鲎试剂进行热原产品会检查失败。
In 2013, the U.S. FDA began to request applicants submitting biologics licensing applications (BLA) to provide evidence that endotoxin spiked into a drug product could be detected using the USP BET. Referring to confidential data from spiking studies submitted in BLAs, the FDA reported in 2014 and 2015 that poor endotoxin recoveries were observed in some cases, which confirmed the initial findings of Chen and Vinther. (1,8,9)The data from endotoxin spike/hold studies presented in BLAs were confounding and difficult to interpret, prompting the FDA to request more information and/or additional studies for clarification. But spike/hold study protocols varied greatly from study to study and company to company. The detection of endotoxin from spiked samples was influenced by sample preparation methods, including pretreatment and vortexing time.The type of spiking standards, the spike amount, hold-time and temperature of the spike sample, endotoxin recovery calculations, and test execution all contributed significant variability to results, making them difficult to interpret and compare. Certain proteins with isoelectric points (pI) above 8.0 or with positively charged conformational regions in the protein structure were thought to be binding spiked endotoxin. Yet, certain protein-free excipient solutions also appeared to affect the detectability of endotoxin by the LAL test.
2013年，FDA开始要求生物制品上市申请（B）申请人提交在药品中加入内毒素标准品使用USP规定的细菌内毒素检查方法可以检出药品中预加入的细菌内毒素。FDA在2014年和2015年的BLAs中递交的数据中可以发现在一些案例中发现低内毒素回收的现象，这佐证了最初的发现。在BLAs中关于内毒素加标试验及内毒素保持时间的研究是令人费解的和难以解释的，这推动FDA需要获取更多信息或额外的研究来说明这一现象。但是不同公司和不同研究的spike/hold study存在明显的差异。加标样品的细菌内毒素的检出受样品的制备方法影响（包括预处理及涡旋时间等）。细菌内毒素标准品的类型、细菌内毒素标准品的加入量、保持时间、被加标样品的温度、内毒素回收率的计算方式、检查的执行等均可引起测试结果的明显差异，这增加了对结果进行比较和分析难度。等电点（I大于8或具有正电结构的特定蛋白质会束缚加入样品中的内毒素标准品从而引起LER。然而，某些不含蛋白质的赋形剂溶液在使用LAL进行内毒素检查时会内毒素的可检测能力。
Due to the complexity of biological drug formats and materials, historical data do not necessarily provide an adequate basis for a safety assessment of new drugs, supporting the need for LER studies in BLAs for specified biologics regulated by CDER-FDA. Subsequently, PDA recruited from its members, academia, FDA, the biopharmaceutical industry, reagent suppliers, testing, and consulting firms to form the LER Task Force in 2016 toaddress these perplexing, contradictory results.
因为生物制品药物的类型和原料的复杂性，历史数据并不能为新药的安全性提供足够的支持，因此在CDER-FDA监管的特定生物制剂的生物制品上市许可申请中需要对LER进行研究是必要的。随后，2016年PDA从其成员、学术机构、FDA、生物制品企业、试剂供应商、测试机构和咨询公司中招募人员组成LER研究小组设法解决
1.1Purpose目的
Members of the PDA LER Task Force prepared this technical report to describe the underlying mechanisms and contributing factors of LER, summarize the potential clinical impact of the LER phenomenon, present guidelines for developing LER hold-time study design, and provide strategies for the mitigation of LER.
PDA的LER研究小组筹备的这份技术报告，描述了LER的作用机制和相关影响因素、L概述了LER现象的潜在临床影响、提出了LER保持时间研究设计的指导方针、提出了减轻LER的策略。
1.2Scope范围
Focusing primarily on protein drug products, this technical report provides an overview of the regulatory expectations for sterile and/or pyrogen-free drug products and recommend how these requirements can be met through the implementation of microbial process control and testing strategies. It includes a standardized LER hold-time protocol that can be used to determine if a biological drug product is affected by LER.Thirteen case studies from pharmaceutical companies, vendors, and consulting firms comprise Section 8.0, offering a snapshot of the complexity and diversity of LER occurrences across various types of products and formulations.Based on these case studies, Section 4.0 seeks to provide a mechanistic understanding of the LER phenomenon wherein the endotoxin is masked.Section 4.3 describes a two-step reaction model of LER for formulations containing a chelating agent (e.g., citrate) and a surfactant (e.g., polysorbate). As proteins may also mask endotoxin resulting in LER, the reversibility of the LER reaction and its effect on endotoxin detectability in the LAL assay is discussed.Examples provide approaches used to detect or unmask endotoxin. Successful mitigation strategies allow for the use of validated in vitro endotoxin testing methods for drug product release and avoid the use of the pyrogen test using rabbits。
本技术报告主要聚焦于蛋白药物产品，提出了对无菌或无热原的药品的监管要求，并提出如何通过微生物的过程控制和检验策略制定来达成这些要求。它包括一个用于确定生物制品药物是否存在LER现象的LER保持时间研究标准方案。源于制药公司、供应商和咨询公司的13个案例构成了第8节，第8节通过对各种不同类型产品或配方中LER现象的复杂性和多样性的快照。基于这些研究，第4节提供了一种机制用于理解在内毒素被遮蔽时出现的LER现象。第4.3节描述了含有螯合剂（如柠檬酸盐）和表面活性剂（如聚山梨酯）的配方中出现两步反应而导致LER现的模型。因蛋白质可能也会因遮蔽内毒素而引起LER，所以对LER的可逆性及其对使用LAL检验时细菌内毒素可检测性的影响进行了讨论。这些例子提供了细菌内毒素检出或避免遮蔽的方法。成功的LER缓解策略使经验证的体外细菌内毒素检查方法可用于产品放行，而避免用热原检查法。
小结：看完介绍明白一点，含有单抗、螯合剂、表面活性剂的生物制品需要进行研究，对于生物制品需要进行低内毒素回收的研究。
