【行业资讯】 PDA TR 80《制药实验室数据完整性管理体系》(中英文对照版)

2018-11-05 16:15:18 / 打印


5.0 Data Integrity in the Pharmaceutical Microbiology Laboratory

5.0 微生物实验室的数据完整性

5.1 General Considerations and Risks


The approaches used to investigate the occurrence of suspected data integrity issues that h recently occurred in a pharmaceutical microbiology laboratory can be challenging and, in some cases, may be very different than those used to evaluate similar occurrences in an analytical chemistry laboratory, Many microbiological methods are performed manually;

subsequently, the recorded results are often based on the visual observations by an individual scientist performing the tests.


Listed below are a few examples of regulatory observations, Warning Letters, or other institutional accounts that note data integrity problems associated with microbiological laboratory records. These are only examples and are not intended to be an all-inclusive list of concerns:


●Company failed to record and report reliable and accurate data for the environmental monitoring (EM) results; for example, contamination in Grade A rooms were recorded and reported as having no viable microorganisms present when, in fact, microbial contamination was present. Specifically, the settle agar plates used in these areas were felsely reported as having “ no colonies present” but were found to contain 16 colony-forming units (CFU), more than could be reasonably overlooked.


●Company failed to implement an adequate quality assurance system as evidenced by: product sterility test failures that occurred and were not reported, investigated, or documented; five batches of viral harvests that were rejected due to contamination,yet no reports were initiated;and a company practice that product sterility test failure(s) were investigated only if more than one test jar per batch of the first or second harvests failed for sterility.


●Company used a contract laboratory to perform microbiological testing; however, the company audit checklist used to evaluate the suitability of this laboratory was completed by the contract laboratory, not the company, with no follow-up verification.


●Private testing laboratory claimed to have conducted microbiological testing, yet it did not have the laboratory equipment (i.e., incubators) and/or media necessary to perform the analysis.


●Company used an “ unofficial” notebook to record microbial contamination in the plant’s water system; however, there was no official investigation or documentation regarding the water system contamination with a known pathogen (Pseudomonas aeruginosa).[Comment: This type of observation relates to both the microbiology laboratory and operators’ behaviors. Periodically, deceptive individuals will use the same technique to mislead, misrepresent, and/or obscure emerging microbial problems in manufacturing equipment that can impact product quality.]


●Company recorded results that the growth promotion test on the media used for simulation studies supported growth meeting the standard set by USP Chapter <71> Sterility Tests when, in fact, the microorganisms did not grow (30). In another case, filamentous fungi were seen growing in all five spiked media tubes, indicating contamination, yet laboratory records claimed that all five distinct species of microorganisms were actually growing per the USP standard.


Again, while this list is not exhaustive, it does present actual inspection observations made by several regulatory auditors during their documentation of data integrity anomalies in pharmaceutical microbiology laboratories.


5.1.1 Interviewing Analysts


One critical element in conducting an audit for data integrity problems in a microbiological laboratory is interviewing the individuals who perform the QA/QC tests, in particular, the laboratory analysts or technicians. When reviewing analytical results reconded on worksheets or data printouts from the LIMS, for example, it is extremely difficult to detect data that should have been recorded but was not. Much of what analysts learn comes from on-the-job training,  yet unofficial dialogue with cowokers or supervisors is rarely captured or documented. For instance, when a senior analyst instructs a junior analyst on how to handle the appearance of “unwanted” microorganisms found growing on analytical petri plates (such as“write the numerical count of the suspected colonies on the lid of the petri dish but don’t record it on the official worksheet until the supervisor has a chance to review it.”),this “unofficial” practice will not be found in the company’s standard operating procedures(SOPs). An auditor can best assess the potential for inappropriate practices, first, by verifying the acceptance criteria described in SOPs and,then,by inquiring of the analysts or technicians if they have been instructed to adjust or modify data or divert from the laboratory SOPs in any form. Without conducting such foce-to-face interviews, this kind of microbiological data manipulation would be extremely difficult to detect.


5.1.2 On-site Laboratory and Sampling Review


There are several procedural steps when handling, shipping» and storing microbiological samples that can dramatically and negatively impact final analytical results. Specific examples of managing samples that can diminish recovery of microbiological or endotoxin results and, thus, should be avoided through procedures and adequate employee training, are listed below:


●Collecting in-process product or water samples in an inappropriate container that may bind (or remove) a bacterial endotoxin from the sample may later reduce the true level of bacterial endotoxins within that test portion.


● The excessive use of disinfectants to spray the sampling port(s) prior to taking the sample may result in the disinfectant dripping into the sample bottle and reducing the actual bioburden of the product sample. Sampling from the sample ports should closely simulate the common practices performed during routine production operations.


● When using in-line sample collection manifolds for collecting intermediate-stage product manufacturing, the manifold needs to cool down after heat sterilization so it does not kill the microbes collected for laboratory testing.


● Product sample collection bags should not be held directly under a UV light within a storage cabinet. Exposure to UV light will kill the microbes present before the sample collection bags are delivered to the laboratory for testing.


● Microbiological sample bags should not be stored in a freezer or extremely cold refrigerator, which may injure or stress indigenous microbes. Microbial samples should be placed within a controlled environment to preclude any harmful or deleterious impact on the samples; then, analyses should commence as soon as reasonably possible.


Finished products or in-process samples that are undergoing sterility testing in a Class 100 clean room (or isolator) should be disinfected using an antimicrobial solution and a validated contact time. Disinfectant vapors or solution passing through the product packaging or container-closure and into or onto the product prior to sample analysis may prevent or kill microbes in the media-enrichment test environment, which may inhibit recovery of product contaminants. For example, if the product sample was contaminated prior to the package disinfection step, a vulnerable container-closure system prepared with excessive disinfectant would likely result in false negative data. The sample container disinfection process should be included in laboratory qualification studies and the suitability tested to ensure that residual disinfection solution does not alter the integrity of the sample results.


Because microbiological results are not captured in a chromatograph, as with HPLC or GC analysis, opportunities to directly review any anomalies between the raw and recorded data are few. One way to verify the accuracy of the test data documentation is to set aside all available microbial petri plates, broths,identification strips,etc., in a secure area or refrigerator just after they have been removed from the incubator and analysis is complete. A careful review of the raw data against the analytical worksheets or LIMS data entries should reveal any discrepancies and provide an opportunity to catch any problems with either personnel training or equipment recording, and thus improve, laboratory procedures.


Another technique may prove effective in checking data from samples stored in refrigerators and freezers. The ordinal test vessels from completed analyses of all positive samples (in-process or finished product bioburden test results, sterility test results, water testing, EM, personnel, etc.) usually contain microbial isolates. Conducting an inventory of the stored samples can provide a starting point to determine if there are differences between what was actually recovered during sample analysis and the data recorded on worksheets or in the LIMS. While taking inventory of samples stored in freezers and refrigerators,be sure to note the product name, lot numbers, tracking numbers, microbial identification, etc., marked on the exterior of test tubes and petri dishes. This collected information will provide a list that can later be compared to the results recorded on batch records, worksheets, and LIMS. The information gathered from reviewing the stored sample plates and tubes can indicate whether a company’SOPs are effective or if the analysts are following procedures correctly when there are objectionable microorganisms that need to be investigated. If the number and types of actual microbial isolates found from the stored samples do not match those in the company’s quality control records, these data integrity issues maybe due to intentional misreporting of the data.


5.1.3 Worksheet Review


Microbiology data often relies on less automated recordkeeping that can be manipulated, often without clear traceability to the original record, when recording forms and worksheets are not well controlled or reconciled. In order to cross-check and confirm data in worksheets, entries in linked documentation should be reviewed (e.g., logbooks of equipment, batch records). Control over the issuance of original paper records, forms or worksheets should provide mechanisms that allow the original records to be distinguished from copies and reconciled. An expert microbiologist recommends reviewing analytical worksheets carefully during an inspection for the following indications of possible falsification: (a) lab reports containing more tests than can reasonably be run per day or per week or by an individual analyst; (b) backlogged sample worksheets reviewed and approved as a batch at the end of the month; (c) worksheets completed by personnel not present in the lab the day of the tests; (d) worksheets reviewed and approved by some other than an authorized reviewer/approver; (e) changes in handwriting signatures; (f) changes in reporting format, e.g” <1 CFU being replaced by zero; (g) results with counts reported as merely meeting specifications; (h) a change in frequency of OOS result reporting; (i) perfectly filled-out worksheets, indicating a risk that they are a second copy; (j) use of media before it passes growth promotion; (k) missing worksheets or worksheets out of numerical order; (l) discrepancies between investigation reports and worksheets; and (m) results from a single batch reported twice (31)»


5.1.4 Contract Laboratories


Companies that do not have the laboratory space, specialized equipment, or capacity to conduct highvolume microbiological analyses may outsource this type of testing to contract laboratories. In such cases, the company is responsible for thoroughly investigating the reputation and quality control performance of any new or unfamiliar contract laboratory with which they engage in a quality agreement. Before signing an agreement, it is recommended that a manufacture(a) confirm that the contract laboratory complies with CGMP regulations regarding laboratory qualifications and can perform the  required compendial testing for microbial recovery and bacterial endotoxins (30,32— 34); (b) conduct an on-site audit; and (c) submit a sample batch for testing to ensure the quality of testing and data integrity. The company may wish to submit a blinded challenge of test samples that should produce OOS results. Documentation that the laboratory management is qualified to interpret microbiological sample results is required, especially if those individuals will be used as subject matter experts for data review or in the event the lab management needs to conduct a laboratory investigation for questionable sample results.


Reports written based on samples tested by a contract laboratory should include complete analytical worksheets from the analysis performed (e.g., raw material, in-process product intermediates, EM sampies, water, finished products). AJ1 of the original data (including calculations) and all of the positive (growth promotion results) and negative controls used during analysis should be included in the response report as they are equally important to the final product analysis results. The full investigation report of any OOS must also be provided. A certificate of analysis with a summary of the analytical results does not provide all of the information needed to verify sample test data or to satisfy a regulatory audit.


Two laboratory areas that the sponsor or owner company must carefully oversee are: (a) monitoring of source data, including electronic data and metadata, at the contract site, and (b) establishing contractual provisions to ensure the contract site does not delay, deny, refuse, or limit the ability of the sponsor or owner company to inspect their source data. As observed by experienced consultants, some contract sites only allow the sponsor company to review printouts, refusing access to their source electronic data and metadata. Risk-based reviews of critical source data and metadata and reconciliation of source data with reported information is essential for a meaningful data integrity audit.

If a company obtains services from more than one contract laboratory~~a common practice, particularly when special laboratory equipment or expertise is needed—it should thoroughly investigate the reputation and quality control performance of each laboratory engaged. Though rare, the possibility exists for a contract laboratory to falsify data or adjust reported analytical results for microbiologically compromised samples, threatening the company’s product and reputation as well as patient safety. Another potential risk is that each contract laboratory uses a different microbial identification platform (e.g., genotypic vs. phenotypic) that may generate different species names for the same isolate. This can make it difficult for investigation of any analytical OOS results to find the true potential source of product contamination with manufacturing EM isolates that may have used yet another type of microbial identification system.


Lending credibility to the concern about the honesty of private testing laboratories is a court case prosecuted by the U.S. Department of Justice for the FDA. In 2008, the FDA brought criminal charges against a private contract testing laboratory that was caught falsifying data for more than 350 sample worksheets. All 350 sample worksheets showed that the laboratory president performed all analytical steps at “ 1PM” each day. The worksheets indicated that the submitted nutritional supplement product samples were analyzed for yeast and mold. In most cases, the analytical results were made available on the same day the samples were received. The FDA investigators became suspicious of the results on the worksheets because the laboratory had none of the reagents, media, or equipment needed to perform these analyses. Tlie unsuspecting manufacturers that sent their products to this contract laboratory and failed to detect that these sample results were fraudulent (25)


A challenge that some license holders or product owners may have when auditing contracted facilities is limited access to records or information that may reflect a widespread systemic problem at the contracted site and may go undetected. These challenges should be anticipated early in the business and quality agreement discussions to ensure sufficient access to the appropriate records, procedures, and systems during record audits, especially when dealing with OOS or data integrity events. A dual responsibility exists to ensure that all products will be produced and tested according to the regulatory requirements, especially when regulators see contracted facilities as an extension of a manufacturing operation.


5.1.5  Equipment and Instrument Review


If not handled correctly, a varietyof equipment and instruments used in a pharmaceutical microbiology laboratory (e.g., incubators, water baths, pH meters, EM devices, temperature monitoring systems, microscopes) can become the source of a data integrity problem; some of these will be discussed in greater detail later in this section. The general concern is that the results derived from the analyses conducted with equipment and instruments not calibrated or not used properly may generate erroneous results. The three examples that follow describe how the review of quality control shortcoming of laboratory or manufacturing equipment can be critical in detecting erroneous results. Lack of quality control could result in overlooking microbiological conditions or contamination that may compromise manufacturing monitoring and valid product testing.

如果没有正确使用,则由于微生物检测设备(例如培养箱,水浴锅,pH计,环境监测设备,温度监控系统,显微镜等)的可变性可能产生数据完整性问题的源头。这些问题会在本节稍后进行详细阐述。通常认为微生物检验的设备未经过计量或未正确使用是产生错误数据的原因。接下来阐述的三个例子便是介绍为什么实验室或生产设备的质量控制缺陷审核对发现错误结果至关重要。缺乏质量控制可能导致忽视微生物条件或污染, 可能会危及生产监测和产品检验的有效性。

When water activity devices are not calibrated or maintained properly, the validity of the analytical data derived comes into question. For example, if the sensing head of a device is not cleaned properly before a subsequent product sample is placed in the chamber, regardless of the reason for the error, the impact on the analytical value of the final product for release or testing may be significantly altered. Whether or not the post-assay data had been manipulated or unintentionally changed, the analytical results may have been compromised, possibly affecting product disposition (good or bad). A full investigation is required to determine the potential impact.


A laboratory’s automated microbial identification system (e.g., Vitek II", Biolog) generally stores a data file of all the microbial isolates it has identified from all product or environmental microbial isolates tested on it. These identification platforms should have tracking numbers that trace the origin of the microbial isolate back to the product batch record, LIMS, OOS, investigation reports, or similar data. These microbial identification records can be used in an audit to verify the accuracy of the associated paper trail should there be any question of transparency.


 The dates for when instruments are unavailabledue to needed repair or calibration are usually maintained in a logbook or digital spreadsheet* Comparing the records of nonftmctioning or absent laboratory equipment that might have been used for analysis against the microbiological worksheets may detect potential data integrity problems. The risk for using uncalibrated or nonfunctioning laboratory equipment is greatest when the workload becomes heavy or when tight deadlines are imposed. On the surface, this may appear to be a quality control issue; however, it can become a data integrity concern depending on the level of disrepair or calibration of the instrument used and the ultimate impact on the analytical results.


5.2 Testing for Environmental Monitoring, Sterility, and Bacterial Endotoxins


5.2.1 Environmental Monitoring Equipment


Some of the equipment used most fiequently in the pharmaceutical microbiology laboratory and manufacturing Facility are environmental monitoring devices used to collect volumes of air or surface contact From specific rooms (e.g., ISO—5 manufacturing areas used for filling sterile products or sterility test suite used to analyze finished drug products labeled as sterile). The neglect or mishandling of EM devices can cause misleading or incorrect analytical results about the microbial presence(bioburden) within the room being monitored.


5.2.2 Air Sampling Devices


In general, an air sampling device requires that a petri dish or a specifically designed plastic strip containing nutrient agar be placed into the device; the instrument is then turned on to draw a specific volume of air into contact with the agar surface. At the end of the air sampling dwell time, the petri dish or strip along with any captured microorganisms are removed from the device and placed in an incubator. Subsequently, the number ofgrowing microorganisms on the media is counted. Examples of where data integrity problems can occur with air sampling devices include:

通常,空气采样装置需要将培养皿或含有营养琼脂的特殊设计的塑料条放入装置中; 然后启动仪器以吸取特定体积的空气与琼脂表面接触。 在空气采样结束后,将带有捕获到的微生物的培养皿或条带从装置中取出并置于培养箱中。 随后,对培养基上生长的微生物进行计数。 空气采样设备可能出现数据完整性问题的示例包括:

Visualization Of the Petrl Dish.Signs of agar impingement and/or colonies growing within the agar medium, caused by the directed air being drawn into the device onto the agar surface,should be visible, ensuring that the agar petri dish was placed into the air handling device during the initial room setup or at another time, when appropriate.


Clogged samplers. Over time, the narrow opening of the EM sampler that Funnels the air directly toward the turning petri dish will become clogged, essentially preventing the full volume ofair to impinge on the agar surface. Though a maintenance or operational anomaly; this directly impacts the data integrity of the microbial evaluation of critical worlt areas. Routine cleaning of the device in that narrow opening is essential to ensure data integrity For this equipment.

采样器堵塞。 随着时间的推移,环境取样装置中将空气直接传到培养皿的EM取样器的狭窄开口将变得堵塞,最终不能使得全部的空气撞击琼脂表面。 虽然是维护或操作异常引起,这将直接影响了关键工作区微生物评估的数据完整性。对设备的狭窄的开口定期进行清洁对于确保数据完整性至关重要

Quality control Logbook. As with other laboratory equipment, recording ifor when any ofthe air sampling devices are defectiveoroutofservice in thequalitycontrollogbookisessential.Thedates during which a piece of equipment is listed as “out of service” would not be found in the sample batch records.


Settle Plates.When settle plates are used to passively monitor the manufacturing or laboratory environment, plates located near surfacts exposed to UV light during monitoring may not be suitable.UV light may kill viable airbome microbial contamination, resulting in questionable results. Settle plates exposed too long become desiccate, and unwrapped settle plates exposed within an isolator during vaporised hydrogen peroxide (VHP)or other decontamination steps become unable on support growth. In either case, potentially viable airbome microbial contamination may not be recovered during exposure.

沉降菌检测皿。当用沉降菌监测皿用于被动监测生产或实验室环境时,沉降碟不应放置于在监测过程中会暴露在紫外光下的位置。紫外线可能会杀死空气中的活微生物,导致结果不可信。暴露太久的沉降碟会变得干燥,并且在蒸发的过氧化氢(VHP)或其他去污步骤期间暴露在隔离器内的未包装的沉降碟会变得不适宜微生物生长。 在以上任何一种情况下,暴露期间的微生物污染可能无法真实还原。

Final counts. Any manipulation or adjustment of final microbial counts without sound scientific justification, such as subtracting colony counts near the edge ofthe petri plates or the numberof bacterial colonies appearing at the beginning time period For air sampling plates, is a concernfor data integrity (Figure 5.2.2-1).

Figure 5.2J-1 Misreading of EM Plates

图5.2.2-1 环境监测计划错误读取

5.2.3 Surface and Personnel Monitoring Equipment


Disinfectants should not be applied to a work surface or on an operator's gloved hands prior to sampling those surfaces using replicate organism detection and counting (RODAC) plates. Application of a disinfectant to a surface designated to be monitored before the test is performed will change the results ofthe assay, making the surface appear to be free ofmicroorganisms or producing a lower bioburden.


The manipulation of procedural requirements can obscure the reality of a contaminated facility or a person’s poor aseptic technique resulting in the manufacture of a drug product under conditions that adulterate the finished product and impact patient health. Data integrity detection may need to occur at microbiological recovery stages long before an analyst opens the incubator door and transfers the tubes and petri dishes for colonies to be counted, otherwise, the data may be compromised and meaningless. In some cases, the surface monitoring is not done correctly, e.g., too short a contact time or performed improperly (for instance, only the finger tips are sampled but the fingers are not correctly rolled over the agar, or fingers are overlapping the plate).


5.2.4 Review of Sterilily Test


The laboratory methods employed to conduct a sterility test on finished sterile pharmaceutical drug products or medical devices are delineated in international pharmacopeial sterility test chapters such as USP <71> (30). There are two analytical choices: (a) passing the aqueous product through brane filter to collect the potential microorganisms recovered from its contents, followed by rinsing to remove residual antimicrobial preservative(s) contained in the product; or (b) adding the product directly into the enrichment media. The essential step before employing these validated compendialprocedures is to run a product suitability test in  the presence of a set of required QC microorganismsto ensure that, under the test conditions,the product ingredients do not interfere with the recoveryand growth of these challenge QC microorganisms. The design of the suitability test establishes the procedures used during routine testing to provide scientific proof that the method will work under routine product-testing conditions. 

无菌制剂或无菌医疗器械的实验室方法在国际药典无菌试验章节中有描述,如USP <71>(30),包含两种可选的分析方法:(a)用隔膜过滤器过滤产品溶液,从产品中回收潜在微生物,并随后漂洗以去除产品中所含的残留抗菌防腐剂;或(b)将产品直接添加到培养基中。最关键的步骤是在应用这些药典的方法前需要进行产品适用性实验确保其适应测试条件,产品的成分不会干扰到QC微生物挑战菌的生长。适用性实验的设计为产品方法在日常产品检测条件下使用提供了科学的依据。

When the sterility test is properly performed as described in the compendia, and after determiningthe product suitability with that method, data integrity problems can result if sample handling and product testing is significantly altered or mismanaged from the approved SOPs.The following are risksfor data integrity breaches in sterility testing:


Before product containers (units) are transferred to the sterility testing suite or HEPA-filtered laminar flow hood, the outside of each container is usually subjected to a disinfection procedure:sprayed or wiped with a sporicidal solution to decontaminate the exterior of the container, preventing surface contamination from being transferred into the testing area. Any overexposure to the disinfection solution may penetrate the package or container and kill potential productcontaminants before the sterility test is performed. This critical analytical step for certain producttypes (e.g., medical devices, combination products), if performed incorrectly, can result in a “ falsenegative” sterility test that would allow the release of a potentially contaminated lot of parenteralor implantable products. This data integrity problem may not be obvious on a laboratory work-sheet or detected in LIMS because the destruction of potential microbial results would have curred before the product was tested.


In a pharmaceutical microbiology laboratory, the preparation of the media and reagents used during sterility testing is an important step, which should be performed carefully to avoid mistakes.For instance, when analyzing products by the “direct inoculation” approach, the accidental use of a lower volume of test medium may create an inhibitory growth condition due to a higher concentration of the product or preservatives in the test medium compared to those levels proven acceptable during the suitability test . The early detection of low volume test tubes can be missed when the tubes are located in the middle of the holding rack.


One observation that has been documented on several FDA Form 483s when the membrane filter is tranferred to the enrichment broth, or when a powdered product is added directlyto the medium,and the membrane filter adheres to the top of the inner portion of the test tube above the liquid medium (Figure 5.2 . 4 - 1) .This problem has been traced to the four-inch forceps rather than the recommended 10- or 12-inch forceps that may not allow the transfer of the membrane filter into the broth.A similar mishap may occur when hygroscopicpowder products adhere to the neck of the inner tube, preventing the product from entering theenrichment media and allowing potential product contaminants to grow(Figure 5.2 . 4 - 2) .A third example, documented during the inspections of contract laboratories performing sterility testing on sterile medical devices, involves ineffectively cutting up long catheters or complex devices, preventing the product from contacting the enrichment broth. The portions of the productprotruding from the liquid brothmay be the regions of the device thatcontain the indigenousmicroorganisms (Figure 5.2 . 4 - 3) .


These examples reflect testing conditions that may have gone unrecorded or unobserved by a reviewing supervisor; however,their occurrence may result in false negative data to the final recorded laboratory result. The prevention of problems in the pharmaceutical microbiology laboratory that affect data integrity may need to be fixed at stages before observational or data entry is recorded on a worksheet or in LIMS.

这些例子反映了审核主管可能未审核到的,未记录或未观察到的测试条件; 然而,它们的发生可能导致最终假阴性实验室结果。制药微生物实验室中影响数据完整性问题的预防可能需要在观察或数据记录在工作表或LIMS之前的各个阶段进行修复。

5.2 .5 Bacterial Endotoxin Testing


The bacterial endotoxin test (BET) described in compendial test chapters is an enzymatic assay that employs the reagent limulus amebocyte lysate (LAL) along with a vial of controlled standard endotoxin(CSE).The CSE serves as as artificial, laboratory-derived source of bacterial endotoxin that can be used to perform the suitability test (inhibition/enhancement test); it is also employed as a positive control to ensure that incubation conditions during product testing have not been compromised.The BET can also be conducted using a gel clot procedure or with the use of a spectrophotometer, employing a modified version of the LAL reagent(34).Each of these reagents requires careful handling andstorage, the directions for which are clearly stated in the products package instructions.

在药典测试章节中描述的细菌内毒素测试(BET)是一种酶测定法,使用试剂鲎阿米巴样细胞裂解物(LAL)以及一小瓶受控标准内毒素(CSE).CSE作为人工从实验室中获取的细菌内毒素,可用于进行适用性试验(抑制/增强试验); 它也可用作阳性对照,以确保产品测试期间的孵育条件不受影响。BET测试也可以使用改良形式的LAL试剂(34)并采用凝胶凝块程序或使用分光光度计执行。上述每一种需要小心处理和储存,在产品包装说明中有明确说明。

To minimize potential data integrity problems with the final laboratory test results,when performing the BET method for finished product testing of pharmaceutical drugs or medical devices,there are a few critical parameters that need to be understood, satisfied, and documented:


During product container storage (refrigeratedor at roomtemperature), when vial or ampoulesamples containing aqueous solutions are delivered to the laboratory,the indigenous bacterialendotoxin may form micelles or attach to the glass or rubber stopper surfaces and move out of thesolution. Unless the laboratory SOP for performing the BET on aqueous products includes a mixing step prior to removing the test aliquot, the detectable level of endotoxin determined by the assay will be underestimated and may inaccurately portray the products endotoxin unit value withinspecifications. Consequently, the final analytical results could be impacted, even when using avalidated and properly controlled assay, depending on the outcome of a formal investigation (35) 

在产品在容器中存储期间(冷藏或在室温下),当将含有水溶液的小瓶或安瓿样品递送到实验室时,固有的细菌内毒素可形成胶束或附着到玻璃/橡胶塞表面,并从溶液中移除。 除非水性产品BET实验室检验SOP中包含了在移除测试等分试样之前的应包含混合步骤,否则测试所得的内毒素水平结果将被低估,最终错误的将产品内毒素单位值描述为符合标准。尽管使用一种已经过验证,且已受用的测试方法,最终仍会影响产品分析结果,具体取决于正式调查的结果(35)

An allowable amount of bacterial endotoxin (as defined in thepharmacopeial product mono graphs) may be present in a drug product based on its dosage, route of administration, and totalamount of endotoxin delivered, e.g., within a one -hour dosing for an injectable product. A well -designed BET method requires that the maximum valid dilution (MVD) be calculated before theassay is performed. If it is not calculated precisely, with the formula variables and the units used inthe MVD formula, an incorrect value may result.The data integrity problem may occur if the cal -cukted MVD value is higher than the true number. That would make it possible to run the assay at a higher product dilution, creating a testing situation where unacceptable levels of endotoxinmaybe present in the product but released without detection.Since endotoxin have dire patient consequences, this is a crucial data integrity matter .

产品中可能存在可允许量的细菌内毒素(如药学产品单图中所定义),基于其剂量,给药途径和存在于药物产品中的内毒素的总量,例如注射剂,一小时的剂量。一个有效的设计较好的BET方法要求在进行检测之前计算最大有效稀释度(MVD)。如果未精确计算,在使用公式变量和MVD公式中使用的单位,可能会导致不正确的值。 如果计算的MVD值高于真实数字,则可能会出现数据完整性问题。 检测可能在较高的产品稀释度下执行,导致产品中实际存在不可接受的内毒素水平,但最终产品未能检测到而被放行。由于内毒素会对病人造成严重的后果,因此这是一个至关重要的数据完整性问题。

In a U.S. Department of Justice criminal prosecution brought by the FDA involving a fraudulentand contaminated hormone product, the drug manufacturer submitted the bacterial endotoxin analytical data as part of the laboratory evidence. The FDA laboratory detected levels of bacterial endotoxin in the vials used for product rehydration above USP specifications.The drug manufactuer employed a private laboratory to test the same suspect lot for bacterial endotoxin andreported the levels of bacterial endotoxin detected as below the compendial specification. Laboratory records showed that the analyst for the private lab manipulated the product dilution to avoiddetecting the actual level of endotoxin in the product.The analytical report had been tailored togive a finished result with an endotoxin level below the USP alowable limit. The FDA laboratory,however, performed the full range of product dilutions toavoid missing the actual endotoxincon-tamination level.The FDA’s test data proved the contract laboratory had manipulated the dilution to produce false results for their client. This example illustrates the need to both understand the analysis procedure and recognize how it can be manipulated.