Abstract

摘要

Visible residue limits (VRLs) have been shown to be a valuable tool in a validated cleaning validation program.

可見殘留限度(VRLs)已經(jīng)成為了清潔驗證過程中一個有價值的工具。

?

Visible residue limits (VRLs) can be quantitatively established for pharmaceutical products, APIs, excipients, and detergents (1-3). The VRL is the lowest level of residue that can be seen by a panel of observers under a defined set of observation parameters. If a VRL is lower than the calculated acceptable cleaning residue limit for a piece of equipment or an equipment train, then the VRL could be considered the cleaning limit for that equipment. The regulatory agencies have accepted the use of VRLs as a valuable tool to help develop, validate, and maintain a robust cleaning validation program (4, 5).

對于藥品、API、輔料和清洗劑，可見殘留限度(VRLs)能夠被量化制定。VRL是殘留物的最低水平，這些殘留物在一套設(shè)定好的檢測參數(shù)下能夠通過檢測儀表設(shè)備能被看到。如果對于一個設(shè)備或設(shè)備組件，VRL低于計算出的接受清潔殘留限度，那么VRL應(yīng)該被認為是設(shè)備的清洗限度。監(jiān)管機構(gòu)已接受使用VRLs作為一個有價值的工具去開發(fā)、驗證和維護一個健全的清洗驗證程序。

?

The ruggedness of VRLs has been established for different residues, personnel and viewing conditions (6), presentation of coupons for personnel observation (7), and for different materials of construction (MOCs) (8). Through all of these studies, the method of preparation of the VRL samples was held constant. A solution or suspension was prepared in a volatile solvent, typically methanol, serial dilutions were made to decreasing concentration levels and coupons were spiked by pipetting 100 μL onto the MOC coupon. The methanol spread out to a circle of about 5-cm diameter and rapidly evaporated, leaving a ring of visible residue.

對不同殘留物、人員、觀察條件和觀察樣本、不同材質(zhì)條件下的VRLs進行了研究。通過這些研究，VRL樣品制備方法一直保持不變。溶液或懸浮液在揮發(fā)性溶劑中制備，代表性的是甲醇，經(jīng)過一系列的稀釋濃度水平逐漸降低，并且取樣片被加入標(biāo)定物，通過加入100μL不同化工材料。這種甲醇在直徑5cm的圓形物上展開并且迅速揮發(fā)、留下一個可見殘留物的圓環(huán)。

?

Figure 1: Measuring full area.

圖1：測量全部區(qū)域

?

Background

背景

?

Table I: Spiking preparation and target concentrations.

表1：標(biāo)的制備和目標(biāo)濃度

The initial work on VRLs (1) started with the 100-μg level with a target of 4 μg/cm2to correlate with the level cited in the Fourman and Mullin article (10). Additional levels at decreasing values were also determined, but a true visual limit was not determined if the lowest level tested was visible and it was sufficiently lower than the acceptable cleaning residue limit (ARL) (i.e., at least two times lower or 50% of the ARL). The initial work surveyed a wide range of APIs, excipients, and formulations over a relatively short period of time to establish a complete database for a cleaning validation program in a clinical manufacturing facility. As applications of VRLs expanded (2) and experience grew, the spiking levels of the residues were lowered until a final set of spike levels were finalized, as shown inTable I.

VRLs最初的工作始于100-μg水平對應(yīng) 4μg/cm2的目標(biāo)。其他低于這個值得水平也被進行了測定，但是真實的可見限度有沒被測定，如果測試的最低水平是可視的并且足夠低于可接受的清潔殘留限度（例如，至少低兩倍或者可接受清洗限度（ARL）的50%）。最初工作調(diào)查APIs、輔料和配方的寬泛范圍，覆蓋一個相對較短的周期去建立一個完整數(shù)據(jù)庫為臨床提供設(shè)施的清洗驗證程序。VRLs應(yīng)用程序擴充和經(jīng)驗的增長，殘留物的標(biāo)定水平在不斷降低直到最后設(shè)定標(biāo)定物水平定案，如表1所示。

?

The average of several hundred VRL determinations was 1.1 μg/cm2(6), which made it a practical and achievable correlation to use the VRL as the ARL, although it was noted that roughly two-thirds of the VRLs were able to be seen at the lowest level spiked onto the coupons. There was a clear indication of the current situation. In addition, the average numbers were a little misleading in that as experience increased in determining the VRL levels, the numbers generated began to skew to lower levels (6). Now, when 1 μg of material is spiked onto a stainless steel coupon and allowed to spread out to a 5-cm diameter circle, it is often still visible under defined conditions of viewing distance, viewing angle, and light level. The area of the sample with a 5-cm diameter is 19.6 cm2(π x (2.5cm)2), making the concentration of the sample 1 μg/20 cm2or 0.05 μg/cm2. This would equate to a swab sample of 1.2 μg using a 25-cm2swab area. Extracting the 1.2-μg sample into solvent for analysis results in a solution concentration well below 1.0 μg/mL.

幾百個VRL測試的平均值是1.1 μg/cm2，VRL作為ARL使用是切合實際和可實現(xiàn)的相關(guān)，雖然值得注意的是，大致三分之二的VRLs能夠被視為在取樣片上被最低水平標(biāo)定。這明確的指出了當(dāng)前的形勢。另外，平均值有一點誤區(qū)，因為在經(jīng)驗較少的情況下測定的VRL水平可能使最低水平歪斜。現(xiàn)在，當(dāng)1 μg物質(zhì)被標(biāo)定到無銹鋼取樣片并且允許在5cm直徑的圓形上展開，它在特定的距離、角度和燈光水平下觀察經(jīng)常仍然是可見。5cm直徑的樣品面積是19.6 cm2(π x (2.5cm)2)，樣品濃度是1 μg/20 cm2或 0.05 μg/cm2。這等同于1.2μg樣品在25-cm2的面積上抽濾。提取1.2μg樣品到溶劑中去分析結(jié)果，溶液濃度遠低于1.0 μg/mL。

?

Table II: Spiking preparation and concentrations.

表2：標(biāo)的制備和濃度

?

?

Using a 1 μg/20 cm2or 0.05 μg/cm2VRL as a cleaning limit is not practical. VRLs established using this approach at this level could result in a practical quantitative contradiction between the VRL and the limit of quantitation/limit of detection (LOQ/LOD) of the analytical test method if the VRL is lower than the LOQ/LOD. Per regulatory guidance, the sensitivity of the analytical method should extend below the cleaning limit to assess the effectiveness of the cleaning procedure. This approach, however, is not possible if the visual limit is lower than the analytical limit.

用1 μg/20 cm2或0.05 μg/cm2 的VRL作為清洗限度是不切合實際。用這個方法建立的VRLs將導(dǎo)致VRL與定量限/檢測限之間的矛盾。按照法規(guī)指南，分析方法的靈敏度必須低于清潔限度，以評估清洗程序的有效性。但是如果可視限度低于分析限度，那么這個方法是不可能的。

?

?

Figure 2: Measuring 'donut' area

圖2：測試環(huán)形面積

?

As long as the VRL is above the LOQ/LOD of the analytical method, this approach to determining VRLs is acceptable. However, as personnel gain experience, the VRLs start trending to lower values and the analytical sensitivity will become an issue. Either an alternative, more practical quantitative approach is necessary, or VRLs generated using this approach can be treated as more qualitative with the assurance that the equipment will be visually soiled long before there is a concern for patient safety.

只要VRL在分析方法的LOQ/LOD以上，這個方法測試VRLs是可以接受的。但是隨著人員經(jīng)驗的增長，VRLs起始趨勢是較低的值并且分析敏感度將變成問題。任何一個選擇，較多的實際定量方法是必須的，或者用這個方法生成VRLs能夠被當(dāng)作設(shè)備被可見污染的較多的定性和保證，在很久之前病人安全問題就對此進行過關(guān)注。

?

Methodology

方法學(xué)

There are several alternatives to the VRL methodology described in the first part of this article, both qualitative and quantitative. A second, qualitative alternative would be to spike residue at the ARL and at some level below the ARL (50% or 25%) using the same spiking procedure as described previously. If the residues are visually detected, then there would be confidence that the residue would be visible on equipment at levels lower than the ARL and, therefore, visual inspection would be appropriate to determine equipment cleanliness. The VRL would be listed as ‘less than’ the lowest residue level tested.

For this study, two additional alternative methods of determining practical, quantitative VRLs were explored. A third approach is to continue to prepare the residue samples in the same manner as described and measure the area of only the residue ring by subtracting the area of the inner circle from the larger circle leaving only the area covered by the residue as shown in Figures 2 and 3 using Equation 1:

[Eq. 1]Ring Area = πr2– πr22= π(r12– r22) = π(r1– r2) (r1– r2)

在這篇文章的第一部分對VRL描述的方法學(xué)有幾個可選方案，定性和定量相結(jié)合。第二個定性選擇是標(biāo)定殘留物在ARL和ARL低一些的水平（50%或25%），使用的標(biāo)定過程如前所述。如果殘留物能夠被可視檢測，那么就有信心能夠檢測到設(shè)備上的低于ARL水平的殘留物，因此，目視檢查也適用于檢測清潔線的設(shè)備。VRL被列為“低于”檢測到的最低殘留物水平。對于這項研究，測定實際定量的VRLs的另外兩個可選方法進行了探索。第三個方法是按照描述持續(xù)制備同樣的殘留物樣品，通過大圓減去內(nèi)部小圓的面積僅留下被覆蓋的面積進行測定殘留物圓環(huán)的面積，殘留物如圖2和圖3所示用公式1：圓環(huán)面積=πr2– πr22= π(r12– r22) = π(r1– r2) (r1– r2)

?

Figure 3: Measuring 'donut' area.

圖三：測定圓環(huán)面積

?

For example, spiking 100 μL of a 10-μg/mL spiking solution results in a residue with an outer diameter of 5 cm and an inner diameter of 4.8 cm, the area of the residue is 1.5 cm2 compared to the area of the 5-cm circle alone, which is 19.6 cm2. For a 1-μg residue sample, this changes the concentration from 0.05 μg/cm2to 0.67 μg/cm2. This would result in a swab sample of 17 μg using a 25-cm2 swab area.

例如：取100 μL 10μg/mL的標(biāo)定溶液結(jié)果是一個外直徑5cm和內(nèi)直徑4.8cm的圓環(huán)的殘留物，圓環(huán)殘留物的面積僅是1.5 cm2，相當(dāng)于直徑5cm的圓，它的面積是19.6 cm2。對于1μg的殘留物樣品，濃度從0.05 μg/cm2變化到了0.67 μg/cm2。這將導(dǎo)致17 μg的抽濾樣品用于25cm2的抽濾面積。

Afourthapproach, which should also be more quantitative, is to generate a residue with a uniform coverage. Because low spike levels such as 1 μg in 100 μL result in a ring residue at larger areas, minimizing the area of the residue can be accomplished by spiking a minimum volume of a higher concentration sample. Spiking 20 μL of a 75 μg/mL spiking solution resulted in a more uniform 1.5-μg residue. The resulting uniform residue spread over a circle with a diameter of 0.8 cm and an area of 0.5 cm2and a concentration of 3 μg/cm2or 75 μg/25 cm2.

第四個方法也是比較偏重定量，是去用統(tǒng)一的覆蓋面形成殘留物。因為像1 μg/ 100 μL的低標(biāo)水平導(dǎo)致圓環(huán)殘留物在較大的面積，較小殘留物的面積能夠通過高濃度樣品小體積的標(biāo)定來實現(xiàn)。取20 μL 75 μg/mL的標(biāo)定溶液導(dǎo)致1.5μg殘留物分布比較均一。由此產(chǎn)生的均勻殘留物遍布在一個直徑0.8cm的圓上，0.5cm2的面積和3 μg/cm2或75 μg/25 cm2濃度。

Figure 4: Measuring uniform area.

圖四：測試均勻面積

Serial dilutions were made and similarly spiked. The additional samples are shown inTable IIandFigure 4. It can be seen inFigure 4that the first three spots are uniform while spots four and five are not uniformly distributed. The VRL can be determined using the lowest uniformly distributed residue from spot three for a VRL of 0.5 μg/cm2. Although spots four and five are visible, they are not uniformly distributed and attempting to determine a VRL would result in the same issue as with theFigure 1determination (i.e., overestimating the area resulting in a lower VRL). This situation of the uniform and non-uniform residues is analogous to the LOQ and LOD of an analytical test method. The third spot can be quantitatively determined, but quantitation of spots four and five is not possible even though they are visible.

系列稀釋制成類似的標(biāo)。額外的樣品在表II和圖四中顯示。在圖四中能夠看到前三個斑點分布比較均勻，而第四、五個分布不均勻。VRL能夠用最低均勻分布殘留進行計算，按照斑點三計算VRL是0.5 μg/cm2。雖然斑點三和斑點四是可見的，但是它們分布分布不均勻，如果試圖計算VRL將導(dǎo)致圖一測定相同的問題（例如，評估過高的面積導(dǎo)致VRL較低）。均勻和不均勻殘留物的情況與LOQ和LOD分析測試方法相似。第三個斑點能夠被定量分析，但是斑點四和五的定量是不可能盡管他們是可見的。

Discussion

討論

Table III: Options for establishing visible residue limits (VRLs).

MeOH is methanol, ARL is acceptable cleaning residue limit, LOQ is limit of quantitation.

表三：建立可見殘留限度的選擇

MeOH是甲醇，ARL是可接受的清潔殘留限度，LOQ是定量限。

Each of the four approaches for VRLs has its advantages as well as drawbacks.?Table III?provides a summary for the different approaches including preparation, level of VRL, and primary issue. Each is a reasonable approach as long as the company defines their process and is aware of the intent and limitations for the approach they have chosen.

四個VRLs的方法每個都有自己的優(yōu)勢和缺點。表III對不同方法包括制備、VRL水平和主要問題進行了總結(jié)。每個方法都是合理的，只要公司規(guī)定自己的工藝過程并且清楚自己所選方法的意圖和局限性。

?

The?initial?approach described in the background section of spiking a known quantity of residue in decreasing amounts onto multiple coupons and then measuring the area of the entire circular dried residue provides assurance that visible residue would be detected on the equipment surfaces well before the residue becomes a safety risk in the subsequent manufactured batch. This determination of a VRL provides a quantitative assessment of the “margin of safety” between the VRL level and the ARL of the spiked API or formulation. This approach serves as a much better training tool in that personnel will get a better impression of the appearance of the residues as they near the VRL, which is a valuable perspective to have, particularly for older cleaned equipment that is subject to other permanent marks on the surfaces. It is also practical in that residue will often appear as rings or water spots on cleaned, dried equipment.

最初描述的方法在標(biāo)定已知殘留物數(shù)量的背景章節(jié)中，在多個取樣片上減少數(shù)量，然后測量整個圓面積的干殘留物，保證可見殘留物能夠在設(shè)備表面可以很好的檢測，之前的殘留物對隨后的提供批次是一個安全的風(fēng)險。VRL的測定在VRL水平和標(biāo)定API或配方的ARL之間提供了“安全邊緣”定量評估。這個方法作為人員很好的培訓(xùn)工具在VRL附近將獲得殘留物外觀更好的印象，這是一個有價值的視角，特別對有其他永久標(biāo)記的較老的清洗設(shè)備表面。在殘留物經(jīng)常出現(xiàn)的圓環(huán)或清洗、干燥設(shè)備的水印也有實用性。

?

The major potential drawback to this approach can be seen in?Table I. If a 1-μg spiked sample is visible, which is often the case, it will have a resulting concentration of 0.05 μg/cm2. Swabbing this sample and extracting into 10 mL of solvent would result in a sample concentration of 0.1 μg/mL. If not already below the LOQ of the analytical method, it would be very near to it for most analytical testing technologies, including the commonly used technologies for testing cleaning samples, such as high performance liquid chromatography (HPLC) and total organic carbon (TOC) analysis. Because the VRL should be considered equivalent to the ARL, it is expected that the test method for cleaning samples should have an LOQ lower than the ARL for the residue. The analytical test method might not be adequately sensitive, or if it is sensitive enough, samples would be in danger of failing if anything is detected in the test.

這個方法主要潛在的缺點在表1中能夠看到。如果1μg標(biāo)定樣品是可視的，通常情況下，它將有一個0.05μg/cm2的最終濃度。擦拭樣品和萃取到10mL的溶劑中將出現(xiàn)0.1 μg/mL的樣品濃度。如果這個值在分析方法的LOQ以下，大多數(shù)分析測定技術(shù)比較接近，包括通用的清潔樣品測定技術(shù)，例如高效液相色譜（HPLC）和總有機碳分析（TOC）。因為VRL被認為等同于ARL，所以希望清洗樣品的測試方法有一個比殘留物ARL更低的LOQ。分析測定方法可能不足夠敏感，或者敏感性足夠，所有東西在測定中都能檢測到，那么樣品面臨失敗的危險。

?

This danger of a potential cleaning failure using this VRL approach is unjustified because the majority of the spiked, dried residue area is not occupied with residue. At these low spiked concentrations, a ring is formed and the residue occupies only a small area around the perimeter of the dried circle. Although this approach to determination of VRLs is efficient and provides an accurate determination of the amount of residue divided by the area of the residue, the residue is not a uniform residue and results in a VRL lower than is practical. A VRL determined this way can be misleading if an attempt is made to relate it to the LOQ of the analytical method. VRLs established using this approach, however, can certainly be compared to the ADE calculated cleaning limit to determine that visual inspection is adequate for cleaning confirmation; and to determine how wide the “margin of safety” is between the visual limit and the cleaning limit.

潛在清洗失敗的危險使用這個VRL方法是不能被論證的，因為大多數(shù)標(biāo)定物、干燥的殘留物面積沒有被殘留物占據(jù)。在低的標(biāo)定濃度，形成圓環(huán)，殘留物僅占據(jù)干燥圓周長附近的小部分面積。雖然這個方法測定VRLs是有效的，并且提供了通過殘留物數(shù)量除以殘留面積得到準確的測定，但是殘留不均一導(dǎo)致VRL低于真實水平。VRL測定把這個方法帶入歧途，但是如果企圖把它和分析方法的LOQ相關(guān)聯(lián)。不管怎樣，用這個方法建立的VRLs與ADE計算出的清潔限度比較確認可視性檢查適用于清洗確認；同時測定可視限度和清洗限度之間的“安全邊界”有多寬。

?

The simplest approach is the?second?one described previously, which is to spike coupons at a single level that is lower (e.g., 50%) than the ARL. If the spiked residue is visible, it provides assurance that visible residue would be detected on the equipment surfaces before the residue became a safety risk in the subsequent manufactured batch. This approach avoids the low level practicality issue and also has the advantage of speed in that samples can be prepared relatively quickly and calculations are not necessary. Fewer coupons would be used per residue, therefore, more residues could be qualified at the same time. This approach might be recommended if a company wished to establish a VRL program in an efficient time frame or quickly determine the limitations of detecting the visible residue using multiple distances and viewing angles. This approach would also be useful for training or qualifying a large group of personnel, such as all equipment washers and inspection personnel. This approach is also practical in that residue will most often appear as rings or water spots on cleaned, dried equipment.

之前描述的第二種方法最簡單，是直接標(biāo)定取樣片在ARL更低的水平（例如50%）。如果標(biāo)定的殘留物是可視的，為可視殘留物在設(shè)備表面被檢測提供保證，以前的殘留物成為了隨后提供批次的一個安全風(fēng)險。這個方法避免了低水平的實際問題并且也有速度的優(yōu)勢，樣品制備比較迅速并且不需要計算。每個殘留使用的取樣片較少，因此，多個殘留物能夠同時定性。如果公司希望建立高效時間結(jié)構(gòu)或迅速測定的VRL程序用多個距離和視角檢測可視殘留物的限制，這個方法可以推薦。這個方法也用于培訓(xùn)或很多人的資格確認，比如所有設(shè)備清洗人員和檢測人員。這種方法也實用于殘留物最常表現(xiàn)為環(huán)狀或清潔干燥設(shè)備上的水印。

?

The drawbacks with this approach are that it provides a qualitative determination only, while not well defining the “margin of safety” between the VRL and the ARL. A minor drawback is if the spiked residue is not visible, one cannot be certain that some level between the spiked level and the ARL might still be visible and additional coupons would have to be spiked until a visual residue is observed. The biggest drawback to a purely qualitative approach is that the visual limit should actually be the cleaning ARL if residue is visible at levels lower than the calculated cleaning ARL. However, if a company states in their strategy that they are looking at VRLs, solely from a qualitative perspective and that the lower spike level provides a level of confidence to ensure that visibly soiled equipment will not be deemed ‘clean,’ and uses the calculated ARLs as their cleaning limit, this should be a viable approach and defensible with the agencies.

這個方法的缺點是它僅提供了定性測試，而不能確定VRL和ARL之間的“安全邊緣”。次要的缺點是如果被標(biāo)定殘留物不是可視的，不能確定ARL和被標(biāo)定的水平之間的水平可能仍然是可見的，額外的取樣片不得不標(biāo)定直到觀察到可視殘留物。對于純定性的方法最大的缺點是如果殘留物是可視的且水平低于計算出的清洗ARL那么可視限度應(yīng)該是實際上的清洗ARL。但是，如果一個公司聲明他們在戰(zhàn)略上看看VRLs，僅從定性角度和較低的標(biāo)定水平提供置信水平去確認被可視殘留物污染的設(shè)備不能認為是清潔的，用計算出的ARLs作為他們的清潔限度，這應(yīng)該是可行的方法和站得住腳的中介。

?

The?third?approach is to use the same spiking technique as the first two approaches, but then measure only the ring area that is covered by the residue. There are several advantages with this strategy. The spiking of the coupons is straightforward, the residues appear as they would on cleaned and dried equipment, and most importantly, this approach results in a more realistic determination of a VRL because it includes only the area covered by the residue. The higher VRL determinations also should be well above an achievable LOQ and LOD for an analytical test method.

第三個方法是用與前兩個方法相同的標(biāo)定技術(shù)，但是僅測量被殘留物覆蓋的圓環(huán)面積。這個方法有幾個優(yōu)勢，取樣片的標(biāo)定是簡單的，殘留物出現(xiàn)在清潔干燥的設(shè)備上，并且最為重要的是這個方法導(dǎo)致VRL更真實的檢測，因為它僅包括了殘留物覆蓋的面積。更高的VRL測試也應(yīng)該在分析測定方法的LOQ和LOD以上。

?

The primary drawback of this approach is the potential inconsistency of the ring thickness and the subsequent subjectivity for determination of the ring thickness. The inside edge of the ring might not be well defined and a somewhat subjective assessment will need to determine the measurement inner edge of the ring. The wider the ring, the lower the resulting concentration of the dried residue. This issue would need to be addressed in a protocol prior to generating official VRL data using this approach.

這個方法最顯著的缺點是圓環(huán)厚度潛在的不一致和圓環(huán)厚度后續(xù)的主觀測量。圓環(huán)的內(nèi)邊可能不能很好的定義和進行主觀的評價來測量圓環(huán)的內(nèi)邊緣。圓環(huán)越寬，干燥的殘留物濃度結(jié)果就越低。在使用這個方法生成官方VRL數(shù)據(jù)之前，這個問題需要在協(xié)議中處理。

?

The objective of the?fourth?approach was to have a uniform residue rather than a ring type residue. Advantages to this approach include the more realistic VRL based only on area covered by the residue as well as the fact that because the smaller residues take up less space and therefore fewer coupons, more residue VRLs can be generated using fewer coupons.

第四種方法的目的是為了有均勻的殘留相對于圓環(huán)類型的殘留。這個方法的優(yōu)勢包括更真實的VRL僅基于殘留物覆蓋的面積，而且事實上，殘留越小占據(jù)的空間越小因此取樣片更少，生成較多的殘留物VRLs有較少的取樣片。

?

Potential disadvantages with this approach include: pipetting very small volumes (20 μL) accurately and the concept of LOQ and LOD of visible residue. At some point, residue would still be visible but the concentration not able to be accurately determined.

這個方法潛在的缺點包括：精確吸取較少的體積（20 μL）和可見殘留物的LOQ和LOD的概念。在某一時刻，殘留物仍然是可見的，但是濃度不能被精確測定。

結(jié)論

不同方法建立的VRLs被提出和討論。四個方法中的每一個都是可行的，只要講清楚整個清洗驗證的主計劃和持續(xù)性應(yīng)用。兩個更精確的定量方法被定義，它們看上去大致相當(dāng)于VRLs，但是每個的實用性只能通過生成的數(shù)據(jù)和并列的對比來評估出更好的。

?