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TestMethod Assessment for Bioburden and Endotoxin
生物負(fù)荷和內(nèi)毒素的檢測方法評(píng)估
Swab Recovery Method Assessment
擦拭回收率方法評(píng)估
An equipment rinse is performed using a solvent that doesnot interfere with recovery. The swabbing technique, although it has a specialadvantage over the rinse sampling method, has the major disadvantage of a lowrecovery of collected bioburden.The reason for low recovery is related to the swab fiber matrix, which hindersthe release of
microbial cells.
設(shè)備淋洗使用不干擾回收的溶劑。擦拭技術(shù)雖然比淋洗取樣法有獨(dú)特的優(yōu)點(diǎn)，但其主要缺點(diǎn)是收集到的生物污染物回收率低。其原因與棉簽纖維基質(zhì)阻礙微生物細(xì)胞釋放有關(guān)。
The recovery study results should be assessed by the QC laboratory when reviewing cleaning validation and routine microbiological data. If the recovery results are low, then an RCF should beapplied to the final test results to compensate forsampling method limitations and to determine if the acceptance criteria weremet.
在審查清潔驗(yàn)證和日常微生物數(shù)據(jù)時(shí)，應(yīng)由QC實(shí)驗(yàn)室對(duì)回收試驗(yàn)結(jié)果進(jìn)行評(píng)估。如果回收率低，則應(yīng)在最終試驗(yàn)結(jié)果中使用回收校正因子RCF，以補(bǔ)償取樣方法的局限性，并確定是否滿足接受標(biāo)準(zhǔn)。
For standard bioburden recovery, after swabbing is complete typically the swab is either streaked onto an agar mediumor transferred into a liquid medium, vortexed for about 30 s and then theliquid sample preparation is tested by pour-plate or membrane filtration method[37].
對(duì)于標(biāo)準(zhǔn)的生物負(fù)荷回收，在擦拭完成后，通常既要將擦拭棉簽劃到瓊脂培養(yǎng)基上又要轉(zhuǎn)移到液體培養(yǎng)基中，離心約30 s，然后通過傾注平板或膜過濾法測試制備好的液體樣品[37]
Usinga commercial swab kit is recommended. There are many differentcommercially available swabs that can be used, especially for surface sampling.Most swab wetting solutions contain an emulsification and neutralizing bufferto neutralize cleaning agents that may inhibit microbial growth. Theemulsification solution extracts the microorganismsfrom the swab material and disperses them into the solution. This allows therecovery of any microorganismsexposed to specific cleaning agents
建議使用商用棉簽。有許多不同的商用棉簽可以使用，特別是表面取樣。大多數(shù)擦拭用潤濕溶液有乳化和中和緩沖液，以中和可能抑制微生物生長的清潔劑。有乳化作用的溶液從棉簽材料中提取微生物并分散到溶液中。這樣就可以回收暴露到特定清潔劑中的任何微生物。
It should be noted that typicalrecovery specifications applied to chemical cleaning validation are not appliedto microbiological recovery. For bioburden limits, a 50% recovery isoutstanding; usually recoveries obtained are around 5%–20%, with some companiesstating only “growth” as the recovery requirement. The bioburden recoveryresults for some materials (porous) can even be less. However, the methoddescribed in this Guide should assist in getting recovery rates greater than50%.
應(yīng)注意，通常用于化學(xué)清潔驗(yàn)證的回收率標(biāo)準(zhǔn)不適用于微生物回收。對(duì)于生物負(fù)荷限度，50%的回收率已經(jīng)是很好的了；通?；厥章试?%-20%左右，有些公司只將“生長”作為回收要求。對(duì)于某些材料（多孔材料）的生物負(fù)荷回收結(jié)果甚至可能更少。然而，本指南中描述的方法應(yīng)有助于使回收率大于50%。
Consider that there is one factoraffecting these recoveries that is normally not an issue in chemical cleaning validation: the survival of the microorganisms.
考慮到有一個(gè)因素會(huì)影響這些回收率，這在化學(xué)清潔驗(yàn)證中通常不是一個(gè)問題：即微生物的存活率。
The following data is required to calculate the limits:
計(jì)算限值需要以下數(shù)據(jù)：
Worst-case product/equipment train combination
產(chǎn)品/設(shè)備鏈的組合的最差情況
Lowest specification for productcontamination (103CFU/g aerobicbacteria, 102 CFU/g molds and yeasts) (solids)[46, 109]
產(chǎn)品污染的最低標(biāo)準(zhǔn)（需氧菌：1000CFU/g，霉菌和酵母菌：100 CFU/g）（固體）【46，109】
10% contribution to the totalcount by the equipment (90% of the contamination coming from unknown sources)
設(shè)備污染占產(chǎn)品總微生物限度的10%（90%的污染物來自未知來源）
0.1 safety factor
0.1安全因子
Per current versions of USP21 [46] and Ph. Eur. [109], the microbial requirements for non-sterilepharmaceutical DPs are:
根據(jù)當(dāng)前版本的USP21[46]和EP[109]，非無菌藥物DPs的微生物要求為：
Control of the total bioburden
總生物負(fù)荷控制
Elimination of USP indicator andobjectionable microorganisms
控制USP控制菌和致病菌
Cultures usually need to be heavilydiluted prior to plating; otherwise, instead of obtaining single colonies thatcan be counted, a so-called “lawn” forms: thousands of colonies lying over eachother. Additionally, plating is the slowest method. Most microorganisms need atleast 24 hours to form visible colonies. The enumeration of colonies on agar plates can be greatly facilitated by using colonycounters.
培養(yǎng)物通常需要在注板前進(jìn)行足夠稀釋；否則，就不會(huì)獲得可計(jì)數(shù)的獨(dú)立菌落，而是形成所謂的“菌苔”：數(shù)千個(gè)菌落相互重疊。另外，注板是最慢的方法。大多數(shù)微生物需要至少24小時(shí)才能形成可見的菌落。使用菌落計(jì)數(shù)器可大大方便瓊脂平板上菌落的計(jì)數(shù)。
To quantify the number of microorganisms in a culture,the microorganism is plated on a Petri dish with growth medium. If the microorganism cellsare efficiently distributed (non-spreaders) on the plate, it is generallyassumed that each microorganism cell will give rise to a single colony or CFU.The colonies are then counted and, based on the known volume of culture that was spread on the plate,the cell concentration calculated.
為了量化培養(yǎng)物中微生物的數(shù)量，將微生物注入有促生長培養(yǎng)基的培養(yǎng)皿上。如果微生物細(xì)胞有效地分布在平板上（非鋪展劑），通常假設(shè)每個(gè)微生物細(xì)胞會(huì)產(chǎn)生一個(gè)菌落或CFU。然后對(duì)菌落進(jìn)行計(jì)數(shù)，并根據(jù)在平板上鋪展的已知培養(yǎng)體積計(jì)算細(xì)胞濃度。
The primary technique in countingcolonies is to count each colony dot once. One approach is to set the Petridish on a grid background and count thecolonies in each grid cell, moving in a methodical pattern through all thecells. Marking counted colonies on the back of the Petri dish can also be ahelpful approach. Generally, you will need to count at least three plates; onlyuse plates containing 30 to 300 colonies to make robust inferences. Plates withcolonies that are too numerous to count or with too few colonies need to bere-plated from a new dilution. Another approach is to use a colony counter for inspection of allplates to ensure a consistent counting background.
菌落計(jì)數(shù)的主要技術(shù)是每個(gè)菌落點(diǎn)計(jì)數(shù)一次。一種方法是將培養(yǎng)皿設(shè)置在網(wǎng)格背景上，計(jì)算每個(gè)網(wǎng)格單元中的菌落數(shù)，在所有的網(wǎng)格中有條不紊地移動(dòng)。在培養(yǎng)皿背面標(biāo)記已計(jì)數(shù)的菌落也是一種有用的方法。一般來說，你需要數(shù)至少三個(gè)平板；只使用含有30到300個(gè)菌落的平板來做出有力的推斷。菌落太多而無法計(jì)數(shù)或菌落太少的平板需要從一個(gè)重新稀釋的溶液中重新進(jìn)行注板。另一種方法是使用菌落計(jì)數(shù)器檢查所有平板，以確保計(jì)數(shù)背景一致。
Endotoxin Surface Sampling
內(nèi)毒素表面取樣
Bacterial endotoxins are typicallydetected from swab and rinse samples using the LAL method. If swabs are used,extraction methods should be developed prior to processing the samples.
細(xì)菌內(nèi)毒素通常使用LAL法從擦拭和淋洗樣品中檢測。如果使用棉簽，應(yīng)在處理樣品之前開發(fā)提取方法。
Equipment swabbing needs to beperformed by qualified personnel, using sterile swabs made from materials thatdo not interfere with the test. There are several types of swabs used tomonitor flat or hard-to-reach surfaces such as the bottom of a tank, O-rings,traps, transfer lines, and U-bends, although rinse samples are much easier totake.
設(shè)備擦拭需要使用由不干擾試驗(yàn)的材料制成的無菌棉簽并由經(jīng)確認(rèn)的人員來實(shí)施，有幾種類型的棉簽用于監(jiān)測平坦或難以觸及的表面，如儲(chǔ)罐底部、O形環(huán)、存水彎、輸送管線和U形彎管，盡管沖洗樣品更容易獲得。
“The Bacterial Endotoxins test can be performed by the kineticturbidimetric, kinetic chromogenic, or gel-clot test methods. However, thekinetic test methods have significant advantages over the gel-clot test.”
細(xì)菌內(nèi)毒素檢查可采用動(dòng)態(tài)濁度法、動(dòng)態(tài)顯色法或凝膠凝塊試驗(yàn)法。然而，動(dòng)態(tài)測試法比凝膠凝塊試驗(yàn)法有顯著優(yōu)勢(shì)。”
“A BET involves analyzing the liquid sample or sample extract using LimulusAmebocyte Lysate (LAL). LAL is a reagent made from the blood of the horseshoecrab. In the presence of bacterial endotoxins, the lysate reacts to form a clotor cause a color change depending on the technique. The test sample is comparedto a standard curve made from known endotoxin concentrations. Alltests are performed in at least duplicate. A positive product control andnegative control are included as part of each assay.”
BET涉及使用鱟試劑（LAL）分析液體樣品或樣品提取物。鱟試劑是一種由鱟血制成的試劑。在存在細(xì)菌內(nèi)毒素的情況下，溶解物會(huì)根據(jù)機(jī)理反應(yīng)形成凝塊或引起顏色變化。將測試樣品與已知內(nèi)毒素濃度制得的標(biāo)準(zhǔn)曲線進(jìn)行比較，雙樣檢測。陽性品對(duì)照和陰性對(duì)照作為每次分析的一部分。”
“It is required to demonstrate that the test sample does not interfere withthe ability to detect endotoxins. This is accomplished with the positiveproduct control (also called the spike recovery) for the kinetic test methods,and with a separate inhibition and enhancement assay for thegel-clot method.” [110]
“需要證明測試樣品不干擾檢測內(nèi)毒素的能力。這可以通過動(dòng)力學(xué)檢測方法的陽性對(duì)照（也稱為加樣回收）和凝膠凝塊法的單獨(dú)抑制和增強(qiáng)試驗(yàn)來實(shí)現(xiàn)。
Microbiological(Virus, Mycoplasma, and TSE) Studies to Support Cleaning Requirements
支持清潔要求的微生物（病毒、支原體和TSE）研究
The challenges for cleaningvalidation in biopharmaceutical and biological processes are somewhat differentfrom the traditional validation of small-molecule chemical residues. In thebiopharmaceutical industry, the main issue is microbiological contamination including viral,mycoplasma, bacteria, fungi, and other biological residues, and almost all of these can be overcome byusing sodium hydroxide and other alkaline solutions in the cleaning process.
生物制藥和生物工藝中清潔驗(yàn)證的挑戰(zhàn)與傳統(tǒng)的小分子化學(xué)殘留驗(yàn)證有所不同。在生物制藥工程中，主要的問題是微生物污染，包括病毒、支原體、細(xì)菌、霉菌和其他生物殘留物，幾乎所有這些都可以通過在清潔過程中使用氫氧化鈉和其他堿性溶液來去除。
Most manufacturing facilitiesoutsource the cleaning method/cycle development activities that involvepathogenic or non-flora type organisms due to the inherent challenges andpossible facility and/or laboratory contamination risks. The data generated from these studies supports thedevelopment and validation of the cleaning cycles including types of cleaningagents used, sampling methods, and other relevant process requirements. It isunderstood that special biological handling, precautions, and containmentcontrols should be implemented when working with the organisms described.
由于固有的挑戰(zhàn)和可能的設(shè)施和/或?qū)嶒?yàn)室污染風(fēng)險(xiǎn)，大多數(shù)制造廠將涉及致病性或非植物性有機(jī)體的清潔方法/程序開發(fā)活動(dòng)外包出去。這些研究產(chǎn)生的數(shù)據(jù)支持清潔程序的開發(fā)和驗(yàn)證，包括使用的清潔劑類型、取樣方法和其他相關(guān)工藝要求。眾所周知，當(dāng)處理上述生物體時(shí)，應(yīng)實(shí)施特殊的生物處理、預(yù)防措施和控制措施
Process development and optimization in upstream processincludes various parts:
上游工藝的工藝開發(fā)和優(yōu)化包括以下幾個(gè)部分：
Cell line development andengineering
細(xì)胞系開發(fā)和工程
Cell clone selection
細(xì)胞克隆選擇
Media and feed development
培養(yǎng)基和供給的開發(fā)
Bioprocess development and scale up
生物工藝開發(fā)和放大
“Reactor design, cell harvesting, process control and the correspondinganalytics can be part of the optimization process as well…. These areas areoptimized individually and focus on a robust generation of a high producttiter, high productivity and defined quality…. [Figure 8.1] schematically presents the different optimization areas andlists the most important parameters.” [111]
“反應(yīng)器設(shè)計(jì)、細(xì)胞播種、過程控制和相應(yīng)的分析也可以是優(yōu)化工藝的一部分…。這些領(lǐng)域被單獨(dú)優(yōu)化，并專注于高產(chǎn)品效價(jià)、高生產(chǎn)率和高質(zhì)量的強(qiáng)勁產(chǎn)品…。[圖8.1]示意性地顯示了不同的優(yōu)化區(qū)域，并列出了最重要的參數(shù)。”[111]
During the laboratory scale studies,the criterion at this stage is to perform the necessary challenges to validatethe removal of specific biological agent (analytics) residues to an acceptablelevel from the process stream equipment. The use of cleaning agents should becontrolled as well as all CPPs, and should be scalable to commercial quantities
在實(shí)驗(yàn)室規(guī)模研究期間，該階段的標(biāo)準(zhǔn)是執(zhí)行必要的挑戰(zhàn)，以驗(yàn)證從工藝流設(shè)備中清除特定生物制劑（分析）殘留物至可接受水平。應(yīng)控制清潔劑的使用以及所有CPP，并應(yīng)可擴(kuò)展到商業(yè)數(shù)量
Following process development, process characterization,process transfer, and set up, GMP production takes place in combination withcleaning validation. Technical data from laboratory scale studies should betransferred to the process development group. When scaling up cleaningprocesses from laboratory scale studies (virus removal, etc.) to process developmentoptimization, it is important to maintain some degree of control of specificcleaning parameters such as cleaning solutionconcentration, cleaning methodology, and other factors [111].
在工藝開發(fā)、工藝特性、工藝轉(zhuǎn)移和建立之后，GMP生產(chǎn)與清潔驗(yàn)證相結(jié)合。實(shí)驗(yàn)室規(guī)模研究的技術(shù)數(shù)據(jù)應(yīng)移交給工藝開發(fā)小組。當(dāng)清潔工藝從實(shí)驗(yàn)室規(guī)模的研究（病毒去除等）到工藝開發(fā)優(yōu)化放大的過程中，保持對(duì)特定清洗參數(shù)（如清洗溶液濃度、清洗方法和其他因素）的某種程度的控制是很重要的[111]。
Figure 8.1: Optimization Areas and Parameters in Upstream Processing
上游工藝的優(yōu)化區(qū)域和參數(shù)
Additional information can be found“A guide to planning your Cleaning Validation Study,” by BioReliance [112].
更多信息可以在BioReliance的“清潔驗(yàn)證研究計(jì)劃指南”中找到[112]。
“Theselection and evaluation of model microorganisms for cleaning validationstudies is a critical part of developing a removal/inactivationprotocol. The selection should consider the type of equipment and raw materialsused in production processes, and the model microorganisms should be knowncontaminants or appropriate related models. For example, bacterial and fungalspecies selected should be representative of environmental, human, and materialsource-derived microbial flora, and should include species of knownantimicrobial resistance. An additional factor to consider for a modelmicroorganism selection is its ability to grow as a high-titer stock in both standard microbiological and cell culture media, and its ease ofdetection in a sensitive and reliable assay…. Typical residualcontaminants that can be important for cleaning validation studies include:
“選擇和評(píng)估用于清潔驗(yàn)證研究的模型微生物是制定去除/滅活方案的關(guān)鍵部分。選擇時(shí)應(yīng)考慮生產(chǎn)過程中使用的設(shè)備和原材料的類型，微生物模型應(yīng)為已知污染物或合適的相關(guān)模型。例如，所選的細(xì)菌和霉菌種屬應(yīng)代表環(huán)境、人體和物料來源的微生物菌屬，并應(yīng)包括已知耐受物種。模型微生物選擇需要考慮的另一個(gè)因素是其在標(biāo)準(zhǔn)微生物培養(yǎng)基和細(xì)胞培養(yǎng)基中作為高效價(jià)培養(yǎng)基生長的能力，并在靈敏和可靠的分析中易于檢測…。清潔驗(yàn)證研究中重要的典型殘留污染物包括：
Host-cell proteins
宿主細(xì)胞蛋白質(zhì)
Lipids
脂類
DNA/host-cell nucleic acid
DNA/宿主細(xì)胞核酸
Endotoxins
內(nèi)毒素
Carbohydrates
碳水化合物
Membrane/chromatography matrix leachables
膜/色譜基質(zhì)浸出物
Detergents
洗滌劑
Viruses
病毒
TSEs
TSEs
Mycoplasmas, bacteria, fungi”
支原體、細(xì)菌、霉菌”
來源：GMP辦公室
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