美国FDA药品质量控制微生物实验室检查指南
美国FDA CGMP检查指导手册
FDA检查员指导手册CP 7356.002：药品生产检查程序目录对现场报告的要求 (35)第一部分背景 (36)第二部分执行 (36)2.1.目的 (36)2.2.策略 (36)2.2.1.对生产企业两年一度的检查（包括重新包装商、合同实验室等） (36)2.2.2.系统性检查 (37)2.2.3.对原料药及制剂生产的系统性检查计划 (38)2.2.3.1.质量系统 (38)2.2.3.2.厂房设施与设备系统 (38)2.2.3.3.物料系统 (38)2.2.3.4.生产系统 (38)2.2.3.5.包装和贴签系统 (38)2.2.3.6.实验室控制系统 (39)2.3.程序管理指导 (39)2.3.1.定义 (39)2.3.1.1.监督性检查 (39)2.3.1.2.达标检查 (40)2.3.1.3.受控状态 (40)2.3.1.4.药品工艺 (40)2.3.1.5.药品生产检查 (41)第三部分检查 (41)3.1.检查活动 (41)3.1.1.总则 (41)3.1.2.检查方法 (42)3.1.2.1.全面性检查的选择 (43)3.1.2.2.简略性检查的选择 (43)3.1.2.3.综合性检查范围 (43)3.1.3.系统性检查范围 (43)3.1.3.1.质量系统 (44)3.1.3.2. 厂房设施与设备系统 (44)3.1.3.3.物料系统 (45)3.1.3.4.生产系统 (46)3.1.3.5.包装和贴签系统 (47)3.1.3.6.实验室控制系统 (48)3.1.4.取样 (49)3.1.5.检查组组成 (49)3.1.6.报告 (49)第四部分分析 (50)第五部分法律性/行政性策略 (50)5.1.质量系统 (51)5.2.厂房设施和设备 (51)5.3.物料系统 (51)5.4.生产系统 (52)5.5.包装和贴签系统 (52)5.6.实验室控制系统 (52)对现场报告的要求作为法律行动的一部分，所有针对因在执行cGMP方面有缺陷而采取的检查，均要向药品评价和研究中心的达标办公室呈交一份现场检查报告(EIR)。
美国微生物检查要求及微生物监控体系
USDA FSIS对实验室质量控制的要求
一、检测方法 二、实验室质量保证体系 三、能力验证 四、人员培训 五、动物种别鉴定 六、仪器设备的维护、校正和验证
USDA FSIS对实验室质量控制的要求——检测方法
沙门氏菌:
沙门氏菌是USDA FSIS官方实验室的重点检 测项目，同时也要求企业实验室实施该项目的 检测。对于生肉连续抽取51个样品检测，阳性 样品不得超过12个；对于熟鸡肉（RTE食品）， 检测样品325克，不得检出。罐装食品不做沙 门氏菌检测。
革 兰 氏 染 色
溶 血 试 验
协 同 溶 血 试 验
小 鼠 致 病 性 试 验
SN标准食品中单核细胞增生李斯氏菌检验程序
检样 前增菌方法 消毒 乳、 乳制品、水产品 等加工制品，25g样品加 225mlMBP 30℃，18~24h 1ml接种到100mlLEB 30℃，18~24h MMA平板或OXA平板 35℃，24~48h 挑取可疑菌落，接种TSA-YE 35℃，18~24h TSB-YE 直接增菌方法 生乳等 未加 工样品 ， 2 5 g 样 品 加 225mlLEB
Half-Fraser肉汤 Half-Fraser肉汤 Fraser肉汤 Fraser肉汤
SN标准 SN标准 GB标准 GB标准
MBP
EB增菌液 EB增菌液 EB增菌液、 EB增菌液、LB1 增菌液 肉汤、LB2肉汤 肉汤、LB2肉汤
MMA琼脂、OXA琼脂 MMA琼脂、OXA琼脂 琼脂 MMA琼脂 MMA琼脂
美国官方机构和青岛局食品实 验室对禽肉及产品微生物检测依据
1、美国官方机构对禽肉及产品微生物检测依据
USDA FSIS：《Microbiology Laboratory Guidebook,MLG》 (3RD Edition,1998) FDA：《Bacteriological Analysis Manual online, MAB》
美国微生物检查要求及微生物监控体系
其m=中3，大M于=1C00）0o个。p/也yg的r有i样g一品h些t数企2不业0超0运4过用-3数2个0理，1统大1计于A技1s0术p00评o个s估/eg其的P偏样ty离品的L数程t0d个度. （。n=13，
克。官方也规定了对与食品接触的台面强制性实施李斯特氏菌 官方检查。要求加工厂实验室可使用国际认可的方法对食品接 触的表面的环境表面进行单核增生李斯特氏、李斯特氏属或类 似李斯特氏其中一种进行检查。但官方确认和调查检验中采用 的方法应为农业部实验室的方法检测单核增生李斯特氏。在从 事调查过程中食品安全局还会使用脉冲凝胶电泳技术，并使用 CDC（疾病控制中心）的数据库进行鉴定。
美官方对肉类及产 品的微生物监控要求
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FDA高纯水验证指南
美国FDA高纯水系统检查指南注释：这份文件是检查员和其他FDA人员的参考资料。
这份文件不约束FDA,不授予任何人任何权力、特权、利益或豁免权。
这份指南主要从微生物方面讨论对用于药物制剂和原料药生产的高纯水系统的检查及评价。
它也涉及了对不同类型系统的设计及一些与这些系统相关问题的检查。
如同其它指南一样，它并不是包含一切的，但是提供了对高纯水系统的检查和评价的背景及指导。
微生物实验室的药物质量控制检查指南（1993，5月）提供了另外的指导。
1、系统设计系统设计的一个基本考虑因素是所生产产品的种类。
对于注射剂关注的是热原，这就需要使用注射用水。
注射用水适用于产品配制，以及成分和生产设备的最终清洗。
蒸馏和反渗透膜过滤是USP中列出的生产注射用水的唯一可接受方法。
但是，在原料药和生物技术及一些国外公司中，超滤由于可以使内毒素量减到最少而用于那些针用原料药中。
对于一些眼科药物如眼用冲洗液和一些吸入产品如吸入的无菌水是有热原规定的，我们希望它们的配制使用注射用水。
但是对于大多数吸入剂和眼科用药，它们的配制中使用纯化水。
纯化水也适用于外用药、化妆品和口服制剂。
设计的另一个考虑因素是系统温度。
我们发现热（65-80℃）系统能自身消毒。
虽然公司采用其它系统的花费可能较低，但维护、检测和潜在问题的花费比能源节省的花费要大得多。
系统是否循环或单向也是设计中的一个重要考虑因素。
明显地，持续流动的水受高水平污染的可能性较低。
一个单向水系统基本上是一个死角。
最后，可能最重要的考虑因素是风险评估或要求的质量水平。
同时也应该认识到不同的产品要求不同质量的水。
注射剂要求无内毒素的高纯水。
外用和口服制剂要求较低纯度的水，并无内毒素要求。
即使对于外用和口服制剂也有许多因素决定了不同质量的水。
比如，抗酸剂中的保护剂并不是总是有效的，因此必须制定严格的微生物限度。
质量管理部门应该评估使用他们系统的水生产的每一个产品，并确定在微生物最敏感的产品基础上建立的微生物纠偏限度。
FDA国外药厂检查指南
GUIDE(1) TO INSPECTIONSOF FOREIGN PHARMACEUTICAL MANUFACTURERS BACKGROUND背景There has been a significant increase in the number of foreign inspections of pharmaceutical manufacturing plants in the past few years. This trend is attributable mainly to the increase in the number of pre-approval inspections although the increase has been noted in other areas such as routine GMP inspections and compliance follow-up activities. Considering the resource-intensive nature of the foreign inspection program, it has become clear that effective and efficient inspectional coverage is crucial to the successful management of the program and that can be achieved only through maintenance of consistency and uniformity of inspection and enforcement activities.在最近几年医药制造厂外检查数量显著增加。
这一趋势主要是由增加的前置审批检查的次数虽然增加了在其他领域如日常GMP检查和合规性的后续活动记录。
FDA微生物检测方法
FDA微生物检测方法菌落总数测定——菌落总数的概念●菌落总数是指在被检样品的单位重量（g）、容积（ml）或表面积（cm2）内，所含能于某种固体培养基上，在一定条件下培养后所生成的菌落的总数。
菌落总数测定——卫生学意义●判定食品被细菌污染的程度及卫生质量。
●及时反映食品加工过程是否符合卫生要求，为被检食品卫生学评价提供依据。
●通常认为，食品中细菌数量越多，则可考虑致病菌污染的可能性越大，菌落总数的多少在一定程度上标志着食品卫生质量的优劣。
FDA BAM 菌落总数测定流程检样xg/mL+9xml稀释液（磷酸盐缓冲液）适当十倍稀释样品选择2~3个连续适宜稀释度各取1mL分别加入灭菌平皿内（每个稀释度做两个平行）每皿内加入适量平板计数琼脂（PCA）35 ℃48 ±2h菌落计数FDA BAM 菌落计数方法●选择25~250CFU之间的菌落进行计数，计算公式如下：N=∑C/（1*n1+0.1*n2）*d●所有平板的菌落数都不足25CFU，报告EAPC/ml（g）为＜25*1/d。
EAPC：estimated aerobic plate count●所有平板的菌落数都超过250CFU，但不足100/cm2，报告EAPC/ml（g）为最接近250CFU 的平板菌落数的估计值，乘以相应的稀释度。
●所有平板的菌落数都超过100/cm2，计算平板的面积（直径为90mm的平板面积为65cm2），估计最高稀释度每cm2的菌落数，乘以相应平板面积作为该稀释度的菌落计数结果，报告EAPC/ml（g）为＞65*100* 1/d。
●无法计数的平板报告LA（Laboratory Accident）。
●最终结果保留前两位有效数字。
按照4舍6入，5是奇进偶不进。
●菌落总数测定几点说明●由于检样中采用30/35℃有氧条件下培养，因而并不是样品中实际的总活菌数，一些特殊营养要求的细菌、厌氧菌、微需氧菌、以及非嗜中温细菌，均难以反映出来。
FDA QC实验室检查指南
GUIDE TO INSPECTIONS OF PHARMACEUTICAL QUALITY CONTROL LABORATORIESNote: This document is reference material for investigators and other FDApersonnel. The document does not bind FDA, and does no confer any rights,privileges, benefits, or immunities for or on any person(s).1. INTRODUCTIONThe pharmaceutical quality control laboratory serves one of the mostimportant functions in pharmaceutical production and control. A significantportion of the CGMP regulations (21 CFR 211) pertain to the quality controllaboratory and product testing. Similar concepts apply to bulk drugs.This inspection guide supplements other inspectional information containedin other agency inspectional guidance documents. For example, ComplianceProgram 7346.832 requiring pre-approval NDA/ANDA inspections containsgeneral instructions to conduct product specific NDA/ANDA inspection auditsto measure compliance with the applications and CGMP requirements. Thisincludes pharmaceutical laboratories used for in-process and finishedproduct testing.2. OBJECTIVEThe specific objective will be spelled out prior to the inspection. Thelaboratory inspection may be limited to specific issues, or the inspectionmay encompass a comprehensive evaluation of the laboratory's compliance withCGMP's. As a minimum, each pharmaceutical quality control laboratory shouldreceive a comprehensive GMP evaluation each two years as part of thestatutory inspection obligation.In general these inspections may include-- the specific methodology which will be used to test a new product-- a complete assessment of laboratory's conformance with GMP's-- a specific aspect of laboratory operations3. INSPECTION PREPARATIONFDA Inspection Guides are based on the team inspection approach and ourinspection of a laboratory is consistent with this concept. As part of oureffort to achieve uniformity and consistency in laboratory inspections, weexpect that complex, highly technical and specialized testing equipment,procedures and data manipulations, as well as scientific laboratoryoperations will be evaluated by an experienced laboratory analyst withspecialized knowledge in such matters.District management makes the final decision regarding the assignment ofpersonnel to inspections. Nevertheless, we expect investigators, analystsand others to work as teams and to advise management when additionalexpertise is required to complete a meaningful inspection.Team members participating in a pre-approval inspection must read and befamiliar with Compliance Program 7346.832, Pre-ApprovalInspections/Investigations. Relevant sections of the NDA or ANDA should be reviewed prior to the inspection; but if the application is not availablefrom any other source, this review will have to be conducted using thecompany's copy of the application.Team members should meet, if possible, prior to the inspection to discussthe approach to the inspection, to define the roles of the team members, and to establish goals for completion of the assignment. Responsibilities fordevelopment of all reports should also be established prior to theinspection. This includes the preparation of the FDA 483.The Center for Drug Evaluation and Research (CDER) may have issueddeficiency letters listing problems that the sponsor must correct prior to the approval of NDA/ANDA's and supplements. The inspection team is expected to review such letters on file at the district office, and they are expected to ask the plant for access to such letters. The team should evaluate thereplies to these letters to assure that the data are accurate and authentic. Complete the inspection even though there has been no response to theseletters or when the response is judged inadequate.4. INSPECTION APPROACHA. GeneralIn addition to the general approach utilized in a drug CGMP inspection, the inspection of a laboratory requires the use of observations of thelaboratory in operation and of the raw laboratory data to evaluatecompliance with CGMP's and to specifically carry out the commitments in an application or DMF. When conducting a comprehensive inspection of alaboratory, all aspects of the laboratory operations will be evaluated.Laboratory records and logs represent a vital source of information thatallows a complete overview of the technical ability of the staff and ofoverall quality control procedures. SOPs should be complete and adequate and the operations of the laboratories should conform to the written procedures. Specifications and analytical procedures should be suitable and, asapplicable, in conformance with application commitments and compendialrequirements.Evaluate raw laboratory data, laboratory procedures and methods, laboratory equipment,including maintenance and calibration, and methods validation data to determine the overall quality of the laboratory operation and the ability to comply with CGMP regulations.Examine chromatograms and spectra for evidence of impurities, poortechnique, or lack of instrument calibration.s use systems that provide for the investigation oflaboratory test failures. These are generally recorded in some type of log. Ask to see results of analyses for lots of product that have failed to meet specifications and review the analysis of lots that have been retested,rejected, or reworked. Evaluate the decision to release lots of product when the laboratory results indicate that the lot failed to meet specifications and determine who released them.B. Pre-ApprovalDocuments relating to the formulation of the product, synthesis of the bulk drug substance, product specifications, analysis of the product, and others are examined during the review process in headquarters. However, thesereviews and evaluations depend on accurate and authentic data that trulyrepresents the product.Pre-approval inspections are designed to determine if the data submitted in an application are authentic and accurate and if the procedures listed inthe application were actually used to produce the data contained in theapplication. Additionally, they are designed to confirm that plants(including the quality control laboratory) are in compliance with CGMPregulations.The analytical sections of drug applications usually contain only testresults and the methods used to obtain them. Sponsors are not required tofile all the test data because such action would require voluminoussubmissions and would often result in filing redundant information. Sponsors may deliberately or unintentionally select and report data showing that adrug is safe and effective and deserves to be approved. The inspection team must decide if there is valid and scientific justification for the failure to report data which demonstrates the product failed to meet itspredetermined specifications.Coordination between headquarters and the field is essential for a complete review of the application and the plant. Experienced investigators andanalysts may contact the review chemist (with appropriate supervisoryconcurrence) when questions concerning specifications and standards arise.Inspections should compare the results of analyses submitted with results of analysis of other batches that may have been produced. Evaluate the methods and note any exceptions to the procedures or equipment actually used fromthose listed in the application and confirm that it is the same methodlisted in the application. The analyst is expected to evaluate rawlaboratory data for tests performed on the test batches (biobatches andclinical batches) and to compare this raw data to the data filed in theapplication.5. FAILURE (OUT-OF-SPECIFICATION) LABORATORY RESULTSEvaluate the company's system to investigate laboratory test failures. These investigations represent a key issue in deciding whether a product may bereleased or rejected and form the basis for retesting, and resampling.In a recent court decision the judge used the term "out-of-specification"(OOS) laboratory result rather than the term "product failure" which is morecommon to FDA investigators and analysts. He ruled that an OOS resultidentified as a laboratory error by a failure investigation or an outliertest. The court provided explicit limitations on the use of outlier testsand these are discussed in a later segment of this document., or overcome by retesting. The court ruled on the use of retesting which is covered in alater segment of this document. is not a product failure. OOS results fall into three categories:-- laboratory error-- non-process related or operator error-- process related or manufacturing process errorA. LABORATORY ERRORSLaboratory errors occur when analysts make mistakes in following the method of analysis, use incorrect standards, and/or simply miscalculate the data. Laboratory errors must be determined through a failure investigation toidentify the cause of the OOS. Once the nature of the OOS result has beenidentified it can be classified into one of the three categories above. The inquiry may vary with the object under investigation.B. LABORATORY INVESTIGATIONSThe exact cause of analyst error or mistake can be difficult to determinespecifically and it is unrealistic to expect that analyst error will always be determined and documented. Nevertheless, a laboratory investigationconsists of more than a retest. The inability to identify an error's cause with confidence affects retesting procedures, not the investigation inquiry required for the initial OOS result.The firm's analyst should follow a written procedure, checking off each step as it is completed during the analytical procedure. We expect laboratorytest data to be recorded directly in notebooks; use of scrap paper and loose paper must be avoided. These common sense measures enhance the accuracy and integrity of data.Review and evaluate the laboratory SOP for product failure investigations. Specific procedures must be followed when single and multiple OOS resultsare investigated. For the single OOS result the investigation should include the following steps and these inquiries must be conducted before there is a retest of the sample:o the analyst conducting the test should report the OOS result to thesupervisoro the analyst and the supervisor should conduct an informal laboratoryinvestigation which addresses the following areas:1. discuss the testing procedure2. discuss the calculation3. examine the instruments4. review the notebooks containing the OOS resultAn alternative means to invalidate an initial OOS result, provided thefailure investigation proves inconclusive, is the "outlier" test. However, specific restrictions must be placed on the use of this test.1. Firms cannot frequently reject results on this basis.2. The USP standards govern its use in specific cases only.3. The test cannot be used for chemical testing results. An initial content uniformity test was OOS followed by a passing retest. The initial OOS result was claimed the result of analyst error based on a statistical evaluation of the data. The court ruled that the use of an outlier test is inappropriate in this case..4. It is never appropriate to utilize outlier tests for a statisticallybased test, i.e., content uniformity and dissolution.Determine if the firm uses an outlier test and evaluate the SOP.Determine that a full scale inquiry has been made for multiple OOS results. This inquiry involves quality control and quality assurance personnel inaddition to laboratory workers to identify exact process or non processrelated errors.When the laboratory investigation is inconclusive (reason for the error is not identified) the firm:1. Cannot conduct 2 retests and base release on average of three tests2. Cannot use outlier test in chemical tests3. Cannot use a re-sample to assume a sampling or preparation error4. Can conduct a retest of different tablets from the same sample when aretest is considered appropriate (see criteria elsewhere)C. FORMAL INVESTIGATIONSFormal investigations extending beyond the laboratory must follow an outline with particular attention to corrective action. The company must:1. State the reason for the investigation2. Provide summation of the process sequences that may have caused theproblem3. Outline corrective actions necessary to save the batch and preventsimilar recurrence4. List other batches and products possibly affected, the results ofinvestigation of these batches and products, and any corrective action.Specifically:o examine other batches of product made by the errant employee or machineo examine other products produced by the errant process or operation5. Preserve the comments and signatures of all production and qualitycontrol personnel who conducted the investigation and approved anyreprocessed material after additional testingD. INVESTIGATION DOCUMENTATIONAnalyst's mistakes, such as undetected calculation errors, should bespecified with particularity and supported by evidence. Investigations along with conclusions reached must be preserved with written documentation that enumerates each step of the investigation. The evaluation, conclusion andcorrective action, if any, should be preserved in an investigation orfailure report and placed into a central file.E. INVESTIGATION TIME FRAMESAll failure investigations should be performed within 20 business days ofthe problem's occurrence and recorded and written into a failure orinvestigation report.6. PRODUCT FAILURESAn OOS laboratory result can be overcome (invalidated) when laboratory error has been documented. However, non-process and process related errorsresulting from operators making mistakes, equipment (other than laboratory equipment) malfunctions, or a manufacturing process that is fundamentallydeficient, such as an improper mixing time, represent product failures.Examine the results of investigations using the guidance in section 5 above and evaluate the decision to release, retest, or rework products.7. RETESTINGEvaluate the company's retesting SOP for compliance with scientificallysound and appropriate procedures. A very important ruling in one recentcourt decision sets forth a procedure to govern the retesting program. This district court ruling provides an excellent guide to use in evaluating some aspects of a pharmaceutical laboratory, but should not be considered as law, regulation or binding legal precedent. The court ruled that a firm shouldhave a predetermined testing procedure and it should consider a point atwhich testing ends and the product is evaluated. If results are notsatisfactory, the product is rejected.Additionally, the company should consider all retest results in the context of the overall record of the product. This includes the history of theproduct. The court ordered a recall of one batch of product on the basis of an initial content uniformity failure and no basis to invalidate the testresult and on a history of content uniformity problems with the product.,type of test performed, and in-process test results. Failing assay results cannot be disregarded simply on the basis of acceptable content uniformity results.The number of retests performed before a firm concludes that an unexplained OOS result is invalid or that a product is unacceptable is a matter ofscientific judgment. The goal of retesting is to isolate OOS results butretesting cannot continue ad infinitum.In the case of nonprocess and process-related errors, retesting is suspect. Because the initial tests are genuine, in these circumstances, additionaltesting alone cannot contribute to product quality. The court acknowledged that some retesting may precede a finding of nonprocess or process-basederrors. Once this determination is made, however, additional retesting for purposes of testing a product into compliance is not acceptable.For example, in the case of content uniformity testing designed to detectvariability in the blend or tablets, failing and non-failing results are not inherently inconsistent and passing results on limited retesting do not rule out the possibility that the batch is not uniform. As part of theinvestigation firms should consider the record of previous batches, sincesimilar or related failures on different batches would be a cause ofconcern.Retesting following an OOS result is ruled appropriate only after thefailure investigation is underway and the failure investigation determines in part whether retesting is appropriate. It is appropriate when analysterror is documented or the review of analyst's work is "inconclusive" , but it is not appropriate for known and undisputed non-process or processrelated errors.The court ruled that retesting:o must be done on the same, not a different sampleo may be done on a second aliquot from the same portion of the sample that was the source of the first aliquoto may be done on a portion of the same larger sample previously collectedfor laboratory purposes8. RESAMPLINGFirms cannot rely on resampling. The court ordered the recall of one batch of product after having concluded that a successful resample result alonecannot invalidate an initial OOS result. to release a product that hasfailed testing and retesting unless the failure investigation disclosesevidence that the original sample is not representative or was improperlyprepared.Evaluate each resampling activity for compliance with this guidance.9. AVERAGING RESULTS OF ANALYSISAveraging can be a rational and valid approach when the object underconsideration is total product assay, but as a general rule this practiceshould be avoided. The court ruled that the firm must recall a batch thatwas released for content uniformity on the basis of averaged test results. because averages hide the variability among individual test results. Thisphenomenon is particularly troubling if testing generates both OOS andpassing individual results which when averaged are within specification.Here, relying on the average figure without examining and explaining theindividual OOS results is highly misleading and unacceptable.Content uniformity and dissolution results never should be averaged toobtain a passing value.In the case of microbiological turbidimetric and plate assays an average is preferred by the USP. In this case, it is good practice to include OOSresults in the average unless an outlier test (microbiological assays)suggests the OOS is an anomaly.10. BLEND SAMPLING AND TESTINGThe laboratory serves a vital function in blend testing which is necessary to increase the likelihood of detecting inferior batches. Blend uniformity testing cannot be waived in favor of total reliance on finished producttesting because finished product testing is limited.One court has ruled that sample size influences ultimate blend test results and that the sample size should resemble the dosage size. Any other practice would blur differences in portions of the blend and defeat the object of the test. If a sample larger than the unit must be taken initially, aliquotswhich resemble the dosage size should be carefully removed for the test,retests, and reserve samples. Obviously, the initial larger sample shouldnot be subjected to any additional mixing or manipulation prior to removing test aliquots as this may obscure non-homogeneity.Multiple individual blend uniformity samples taken from different areascannot be composited. However when variation testing is not the object ofassay testing, compositing is permitted.If firms sample product from sites other than the blender, they mustdemonstrate through validation that their sampling technique isrepresentative of all portions and concentrations of the blend. This means that the samples must be representative of those sites that might beproblems; e.g. weak or hot spots in the blend.11. MICROBIOLOGICALThe review of microbiological data on applicable dosage forms is bestperformed by the microbiologist (analyst). Data that should be reviewedinclude preservative effectiveness testing, bioburden data, and productspecific microbiological testing and methods.Review bioburden (before filtration and/or sterilization) from both anendotoxin and sterility perspective. For drug substance labs evaluatemethods validation and raw data for sterility, endotoxin testing,environmental monitoring, and filter and filtration validation. Also,evaluate the methods used to test and establish bioburdens.Refer to the Microbiological Inspection Guide for additional informationconcerning the inspection of microbiological laboratories.12. SAMPLINGSamples will be collected on pre-approval inspections. Follow the sampling guidelines in CP 7346.832, Part III, pages 5 and 6.13. LABORATORY RECORDS AND DOCUMENTATIONReview personal analytical notebooks kept by the analysts in the laboratory and compare them with the worksheets and general lab notebooks and records. Be prepared to examine all records and worksheets for accuracy andauthenticity and to verify that raw data are retained to support theconclusions found in laboratory results.Review laboratory logs for the sequence of analysis versus the sequence of manufacturing dates. Test dates should correspond to the dates when thesample should have been in the laboratory. If there is a computer data base, determine the protocols for making changes to the data. There should be an audit trail for changes to data.We expect raw laboratory data to be maintained in bound, (not loose or scrap sheets of paper), books or on analytical sheets for which there isaccountability, such as prenumbered sheets. For most of those manufacturers which had duplicate sets of records or "raw data", non-numbered loose sheets of paper were employed. Some companies use discs or tapes as raw data andfor the storage of data. Such systems have also been accepted provided they have been defined (with raw data identified) and validated.Carefully examine and evaluate laboratory logs, worksheets and other records containing the raw data such as weighings, dilutions, the condition ofinstruments, and calculations. Note whether raw data are missing, if records have been rewritten, or if correction fluid has been used to conceal errors. Results should not be changed without explanation. Cross reference the data that has been corrected to authenticate it. Products cannot be "tested into compliance" by arbitrarily labeling out-of-specification lab results as"laboratory errors" without an investigation resulting in scientificallyvalid criteria.Test results should not have been transcribed without retention of theoriginal records, nor should test results be recorded selectively. Forexample, investigations have uncovered the use of loose sheets of paper with subsequent selective transcriptions of good data to analyst worksheetsand/or workbooks. Absorbance values and calculations have even been found on desk calendars.Cut charts with injections missing, deletion of files in direct data entry systems, indirect data entry without verification, and changes tocomputerized programs to override program features should be carefullyexamined. These practices raise questions about the overall quality of data.The firm should have a written explanation when injections, particularlyfrom a series are missing from the official work-sheets or from files andare included among the raw data. Multiple injections recorded should be in consecutive files with consecutive injection times recorded. Expect to see written justification for the deletion of all files.Determine the adequacy of the firm's procedures to ensure that all validlaboratory data are considered by the firm in their determination ofacceptability of components, in-process, finished product, and retainedstability samples. Laboratory logs and documents when cross referenced may show that data has been discarded by company officials who decided torelease the product without a satisfactory explanation of the resultsshowing the product fails to meet the specifications. Evaluate thejustification for disregarding test results that show the product failed to meet specifications.14. LABORATORY STANDARD SOLUTIONSAscertain that suitable standards are being used (i.e. in-date, storedproperly). Check for the reuse of stock solutions without assuring theirstability. Stock solutions are frequently stored in the laboratoryrefrigerator. Examine the laboratory refrigerators for these solutions and when found check for appropriate identification. Review records of standard solution preparation to assure complete and accurate documentation. It ishighly unlikely that a firm can "accurately and consistently weigh" to the same microgram. Therefore data showing this level of standardization orpattern is suspect and should be carefully investigated.15. METHODS VALIDATIONInformation regarding the validation of methods should be carefullyevaluated for completeness, accuracy and reliability. In particular, if acompendial method exists, but the firm chooses to use an alternate methodinstead, they must compare the two and demonstrate that the in-house method is equivalent or superior to the official procedure. For compendial methods firms must demonstrate that the method works under the actual conditions of use.Methods can be validated in a number of ways. Methods appearing in the USP are considered validated and they are considered validated if part of anapproved ANDA. Also a company can conduct a validation study on theirmethod. System suitability data alone is insufficient for and does notconstitute method validation.In the review of method validation data, it is expected that data forrepetitive testing be consistent and that the varying concentrations of test solutions provide linear results. Many assay and impurity tests are nowHPLC, and it is expected that the precision of these assays be equal or less than the RSD's for system suitability testing. The analytical performanceparameters listed in the USP XXII, <1225>, under the heading of Validation of Compendial Methods, can be used as a guide for determining the analytical parameters (e.g., accuracy, precision, linearity, ruggedness, etc.) needed to validate the method.16. EQUIPMENTLaboratory equipment usage, maintenance, calibration logs, repair records, and maintenance SOPs also should be examined. The existence of the equipment specified in the analytical methods should be confirmed and its conditionnoted. Verify that the equipment was present and in good working order atthe time the batches were analyzed. Determine whether equipment is beingused properly.In addition, verify that the equipment in any application was in goodworking order when it was listed as used to produce clinical or biobatches. One would have to suspect the data that are generated from a piece ofequipment that is known to be defective. Therefore, continuing to use andrelease product on the basis of such equipment represents a seriousviolation of CGMP's.17. RAW MATERIAL TESTINGSome inspections include the coverage of the manufacturer of the drugsubstance. The safety and efficacy of the finished dosage form is largelydependent on the purity and quality of the bulk active drug substance.Examine the raw data reflecting the analysis of the drug substance including purity tests, charts, etc.Check the impurity profiles of the BPC used in the biobatch and clinicalproduction batches to determine if it is the same as that being used tomanufacture full scale production batches. Determine if the manufacturer has a program to audit the certificate of analysis of the BPC, and, if so, check the results of these tests. Report findings where there is substantialdifference in impurity profiles and other test results.Some older compendial methods may not be capable of detecting impurities as necessary to enable the control of the manufacturing process, and newermethods have been developed to test these products. Such methods must bevalidated to ensure that they are adequate for analytical purposes in thecontrol and validation of the BPC manufacturing process. The drug substance manufacturer must have complete knowledge of the manufacturing process and the potential impurities that may appear in the drug substance. Theseimpurities cannot be evaluated without a suitable method and one that hasbeen validated.Physical tests such as particle size for raw materials, adhesion tests for patches, and extrusion tests for syringes are essential tests to assureconsistent operation of the production and control system and to assurequality and efficacy. Some of these tests are filed in applications andothers may be established by the protocols used to manufacture the product. The validation of methods for such tests are as important as the test forchemical attributes.Physical properties tests often require the use of unique equipment andprotocols. These tests may not be reproducible in other laboratories,therefore, on site evaluation is essential.。
FDA微生物检测方法
FDA微生物检测方法菌落总数测定——菌落总数的概念●菌落总数是指在被检样品的单位重量（g）、容积（ml）或表面积（cm2）内，所含能于某种固体培养基上，在一定条件下培养后所生成的菌落的总数。
菌落总数测定——卫生学意义●判定食品被细菌污染的程度及卫生质量。
●及时反映食品加工过程是否符合卫生要求，为被检食品卫生学评价提供依据。
●通常认为，食品中细菌数量越多，则可考虑致病菌污染的可能性越大，菌落总数的多少在一定程度上标志着食品卫生质量的优劣。
FDA BAM 菌落总数测定流程检样xg/mL+9xml稀释液（磷酸盐缓冲液）适当十倍稀释样品选择2~3个连续适宜稀释度各取1mL分别加入灭菌平皿内（每个稀释度做两个平行）每皿内加入适量平板计数琼脂（PCA）35 ℃48 ±2h菌落计数FDA BAM 菌落计数方法●选择25~250CFU之间的菌落进行计数，计算公式如下：N=∑C/（1*n1+0.1*n2）*d●所有平板的菌落数都不足25CFU，报告EAPC/ml（g）为＜25*1/d。
EAPC：estimated aerobic plate count●所有平板的菌落数都超过250CFU，但不足100/cm2，报告EAPC/ml（g）为最接近250CFU 的平板菌落数的估计值，乘以相应的稀释度。
●所有平板的菌落数都超过100/cm2，计算平板的面积（直径为90mm的平板面积为65cm2），估计最高稀释度每cm2的菌落数，乘以相应平板面积作为该稀释度的菌落计数结果，报告EAPC/ml（g）为＞65*100* 1/d。
●无法计数的平板报告LA（Laboratory Accident）。
●最终结果保留前两位有效数字。
按照4舍6入，5是奇进偶不进。
●菌落总数测定几点说明●由于检样中采用30/35℃有氧条件下培养，因而并不是样品中实际的总活菌数，一些特殊营养要求的细菌、厌氧菌、微需氧菌、以及非嗜中温细菌，均难以反映出来。
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美国FDA药品质量控制微生物实验室检查指南1993年I．导言《药品质量控制实验室检查指南》主要涉及许多有关药品实验室分析的化学方面的问题，对微生物实验室的检查仅提供了有限的指导，而本指南则是微生物分析检查过程的指导。
本指南建议，如同对任何实验室检查—样，在检查微生物实验室时，应有—名熟悉检验的分析学家(微生物学家)参与。
II．非无菌药品的微生物检验由于种种原因，局部药品、滴鼻剂和吸入剂存在许多微生物污染方面的问题.美国药典‘‘微生物属性”章(1111)指出“应该从药品用法、药品性质及时患者的潜在危害等方面评价微生物在非无菌药品中的重要性”，除此之外，没有提供具体的指导。
美国药典还建议应对某些种类的非无菌药品做常规的总菌数检验及某些特定的污染指示微生物的检验：例如：植物、动物和某些矿物质中的沙门氏菌属；口服液体中的大肠杆菌；局部用药品小的金黄色倘萄球菌和绿脓杆菌污染；以及：自肠、尿道、阴道用药中的酵母菌和霉菌：大量的专题文章还沦及厂微生物的限度。
作为非无菌药品受微生物污染的可接受程度和类型的—般性指导，美国食品和药品管理局药品分局的邓尼根博士曾强调其对健康的危害问题。
1970年他提出被革兰氏阴性细菌污染的局部制剂可能引起中度至重度的健康危害：文献和调查表明，许多感染都源于这种局部药品的革兰氏阴性细菌污染。
几年前马萨诸塞州的—家医院就报道过—宗络合碘(Povidonelodine)被洋葱假恤孢菌(Pseudomonas cepacia)污染的典型病例。
因此，每家公司都希望为自己的非无菌药品制订出一种关于微生物限度的标准，美国药典中“微生物限度”(USP61)提供了检验几种指示微生物的方法，但并末涉及所有有害微生物。
例如医药界普遍认为，洋葱假中孢菌在局部药品或滴鼻剂斗，大量存在是有害的，但美国药典没有提供证明这种微生物存在的检验方法。
间羟异丙肾上腺素硫酸盐吸入剂溶液的收回就是这方面的—个例子。
美国药典第XⅫ版各论部分没有要求对这种药品进行微生物检验。
药品管理局将其列为一级收回，因为此药受到了洋葱假单孢菌污染。
健康危害评估表明，这种微生物对肺部感染的风险很大，特别是对某些患有慢性气管阻塞、囊性纤维变性和免疫缺陷等病的患者具有潜在的生命危险。
此外，药典的微生物限度部分所描述的检验程度不能鉴别这些微生物。
现行美国药典在“微生物限度”部分(61)做了复检的规定，但是许多建议要求取消这种复检条款。
类似于其他检验，初次检验结果应予以审查与研究。
微生物污染并不是均匀地分布在一批药品或样品中的。
如果在一个样品中发现污染而在另—次取样样品中没有发现，则不应忽视初次发现的结果。
复检的结果应予以审查与评估，并且应特别注意进行复检的逻辑性和合理性。
为了分离出特定的微生物污染物，FDA实验室和制药工业的许多实验室，使用某些自钝化剂如吐温或卵磷脂的营养培养基，这对于钝化药品中常有的防腐剂的作用是非常必要的，还能为受损的或生长缓慢的微生物提供更好的基质；其他生长参数包括降低培养温度以及延长培养时间(至少5天)，因为它们可以为这类微生物提供良好的生存条件。
例如：在《细菌学分析手册》(BAM)第Ⅵ版中，FDA实验室采用化妆品的检验方法鉴别非无菌药品中的污染物。
这项检验包括将样品置于改良Letheen肉汤中培养。
培养结束后，再用血琼脂平皿和麦球凯琼脂平皿进一步鉴定，然后再鉴别分离出的菌落。
FDA微生物学家用这种方法使所有潜在的病原体的复活达到最佳，并且测定复活的微生物数量及其类别。
FDA分析学家使用这种方法的另—个重要方面就是要确定所有用过的培养暴促进微生物生长的性能。
选择适当的中和剂很大程度上取决于防腐剂的性质和被评价药品所智的配方：如果在营养肉汤出现微生物生长，那么下一步的鉴别就可将样品转到更有选择性的琼脂培养基或适当的增菌琼脂上。
微生物检验可包括对在需氧菌总数检验小发现的菌落的鉴定。
另外上述鉴定不应仅限于美国药典中的指示微生物。
鉴别从微生物总数检验或(和)富集培养检验中分离的各种菌落的重要性，将取决于药品的种类及共其途。
很明显，如果检验—个口服固体制剂如片剂，当检验显示微生物污染量很大时，就可以考虑对分离出的菌落进行鉴别，然而对其他被微生物污染后易引起危害的药品，如局部用药品、吸人剂、滴鼻剂，应鉴别从前落计数及富集检验中分离的菌落。
III．设备、器材和培养基首先审查正在进行的分析，并检查培养中的培养皿．和试管(操作过程中不要疏忽大意而污染检验中的培养皿和试管)。
特别注意那些没有文字记录的复检和经凋查已经鉴别出有污染问题的“特殊项目”。
这种评价可以通过审查正在进行的分析是否有阳性结果来实现。
如有可能要审查前一天的培养皿和培养基，还要把观察结果与实验记录本中的记录进行比较。
检查用于培养基灭菌的高压蒸汽灭菌器。
高压蒸汽灭菌器可能缺少以无菌过滤的空气来代替蒸汽的能力。
对于密封的培养瓶，不会出现这种问题；然而对于不密封的培养瓶，非无菌的空气会导致培养基的污染。
此外，如高压灭菌的时间少于要求的时间，也会使培养基中的污染物生长，并得出错误的阳性结果。
在任务繁重的实验室里，这些问题可能更容易出现。
要检查高压蒸汽灭菌器的温度，温度过高会使必需的营养素变性甚至炭化，这可能使受到抑制的微生物达不到最佳复活。
潜在的假阳性结果带来的明显问题，是无法区别因疏忽大意而产生的培养基污染和直接来源于被检验样品的真正的污染。
Ⅳ。
无菌检验1991年10月11 日，药品管理局颁布了关于经无菌加工艺和最终灭菌生产药品的规定，并列出—份经无菌加工工艺生产的受污染或潜在污染而致收回的药品清单。
许多对收回药品的调查或检查都起因于初次无菌检验不合格。
FDA对生产厂的生产、质控、调查及其缺陷进行中核的结果以及最初无菌检验不合格的证据最终导致厂对这些药品的收回。
美国药典规定，用于进行无菌检验的设施与用于制药的设施应该是一致的，并规定，用于无菌检验的设施不应比无菌加工生产设施造成更多的微生物污染机率。
因此，合理的设计应该包括更衣区域和通过气闸。
环境的监测和着装应该与药品生产中相一致。
由于无菌检验中包括大量药品和培养基的操作，因此检查应包括对无菌检验的实地观察；即使有些公司曾借口实地检查可能会使分析人员紧张而试图阻止，检查组应注意此问题，使实地观察能以尽可能不干扰正常操作环境的方式进行。
然而这种考虑不足以成为放弃这部分检查的充分理由。
检查无菌检验程序最重要的方面之一就是审查元菌检验初次阳性结果的记录。
要求列出不合格的检验结果以便于审查生产、质控记录和调查报告，特别是对于风险性很大的无菌分装的药品，应审查无菌检验初次阳性结果和调查结果。
对于生产厂来说，如果无菌分装的产品在初次无菌检验时出现阳性结果，但无菌检验中的对照未发现异常，则很难决定是否发放该种产品。
应检查是否设立阴性对照品，设立阴性对照对高质量的无菌检验儿为重要；无菌检验规范的做法包括使用已知的最终灭菌或经辐射灭菌的样品作为系统对照，或者使用培养基模拟分装试验中分装的安瓿或西林瓶。
要特别注意生产无菌分装药品的厂家从未发现初次阳性无菌检验结果的情况，尽管这种情况可能发生，但毕竟少见。
曾经有一个案例，某生产厂的记录表明没有阳性结果，实际上他们的记录被改动了。
不存在初次阳性结果，还说明这项检查未通过验证来显示有没有来自于药品或防腐剂的残留抑制物的存在。
检查自动化无菌检验系统或无菌隔离技术，如用于无菌检验的La Calhene装置，这些装置保证了不需要人直接移取样品。
如果被检样品在此系统中检验发现初次检验不合格，很难根据—次复检决定药品是否发放，特别是检验对照是阴性的时候。
评价无菌检验样品的培养时间。
该问题已被阐明，美国药典提出样品至少需要培养7天，而—项提议要求美国药典将其改为14天；希望诸位能评价—项具体的分析过程和产品以确定其最适培养时间。
7天可能不够充分，特别是发现有生长过程缓慢的微生物存在时，培养基灌装、环境、无菌检验结果和其他数据应加以审查以确保没有发现生长缓慢的微生物。
此外，还应比较各种培养方法，以确定它们是否符合已被批准或将被批准的申请书中所列的方法。
V．方法和检验程序的验证确定检验程序的来源。
生产厂的检验程序有几个方面的来源，如美国药典、《细菌学分析手册》及其他微生物参考资料。
事实上对各种有害微生物进行的检验程序不可能得到全面验证，但是应确保样品中的抑制物被中和。
在检查过程中，包括批准前的检查中，要评价微生物检验的方法。
例如我们希望检验方法可以鉴别一些微生物的存在，如洋葱假单孢菌或其他有害的假单孢菌属。
在进行批准前检查时，要将正在使用的方法和申请中呈递的方法进行比较，同时证实实验室拥有必要的检验设备，这些设备在进行关键检验时确实可用并处于良好的运行状态。
美国药典规定，如果已经过正确的验证，另一种方法可以得出同样甚至更好的结果，它可以用来代替法定检验方法。
你也许会发现人们已使用脱水的培养基来制备培养基。
良好的操作包括定期用极少量微生物去测试制备好的培养基，包括使用美国药典规定的指示微生物和正常菌群。
培养基促进生长的能力可能受到培养基制备程序、灭菌过程(过热)和储存的影响，它们是对任何实验室进行检查的重要指标，也是实现微生物实验室优良管理的重要考虑事项。
Ⅵ．数据储存评价检验结果是记录在实验记录本(logbooks)上还是记录在活页检验单上，由于有些生产厂可能不愿提供表明微生物检验结果的表格、总结或打印稿，应当审查数据来鉴别生产过程中潜在的微生物。
当得不到此数据的总结时，检验组应审查充分的数据来建立他们自己对于实验室结果和质量控制程序的总结。
一些实验室将事先印好的表格用于记录检验数据。
有些实验室还指出在检查中审查微生物检验数据的唯一途径是审查单批记录。
然而在大多数情况下，事先印出的表格都是一式几份，而第二，三份总是存在中心档案中。
一些公司以实验记录本记录资料，这些实验记录本也应该被查阅。
另外，很多生产厂装备厂自动化微生物系统，如用于鉴别微生物的Vitek装置。
这种检验的记录和对样品来源的鉴别对于识别生产过程中潜在的微土物问题具有同样价值。
鉴别微生物的自动化系统比较普遍地用于制造非肠道药品的生产厂，用于对从环境、水系统、验证过程和人员的分离物的常规鉴别。
我们巴尔的摩地区的微生物学家都是自动化微生物分析系统使用方面的专家。
他们最先在FDA实验室里使用这种设备并且对于验证这些设备的方法有丰富的经验。
关于该系统的情况或有关问题，可以向巴尔的摩地区实验室查询。
另外，大规模使用这类设备的应当接受来自巴尔的摩地区实验室专家的检查。
Ⅶ．管理审查微生物检验结果是数据评价及解释中一个较难的领域。
评价微生物检验结果需要受过微生物学的专业训练并具备经验。
要厂解检验的方法，而且更重要及更难的是要厂解检验的局限性。