Abstract

摘要

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

可见残留限度(VRLs)已经成为了清洁验证过程中一个有价值的工具。

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).

对于药品、API、辅料和清洗剂，可见残留限度(VRLs)能够被量化制定。VRL是残留物的最低水平，这些残留物在一套设定好的检测参数下能够通过检测仪表设备能被看到。如果对于一个设备或设备组件，VRL低于计算出的接受清洁残留限度，那么VRL应该被认为是设备的清洗限度。监管机构已接受使用VRLs作为一个有价值的工具去开发、验证和维护一个健全的清洗验证程序。

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.

对不同残留物、人员、观察条件和观察样本、不同材质条件下的VRLs进行了研究。通过这些研究，VRL样品制备方法一直保持不变。溶液或悬浮液在挥发性溶剂中制备，代表性的是甲醇，经过一系列的稀释浓度水平逐渐降低，并且取样片被加入标定物，通过加入100µL不同化工材料。这种甲醇在直径5cm的圆形物上展开并且迅速挥发、留下一个可见残留物的圆环。

Figure 1: Measuring full area.

图1：测量全部区域

Background

背景

Table I: Spiking preparation and target concentrations.

表1：标的制备和目标浓度

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水平对应 4µg/cm2的目标。其他低于这个值得水平也被进行了测定，但是真实的可见限度有没被测定，如果测试的最低水平是可视的并且足够低于可接受的清洁残留限度（例如，至少低两倍或者可接受清洗限度（ARL）的50%）。最初工作调查APIs、辅料和配方的宽泛范围，覆盖一个相对较短的周期去建立一个完整数据库为临床提供设施的清洗验证程序。VRLs应用程序扩充和经验的增长，残留物的标定水平在不断降低直到最后设定标定物水平定案，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.

几百个VRL测试的平均值是1.1 µg/cm2，VRL作为ARL使用是切合实际和可实现的相关，虽然值得注意的是，大致三分之二的VRLs能够被视为在取样片上被最低水平标定。这明确的指出了当前的形势。另外，平均值有一点误区，因为在经验较少的情况下测定的VRL水平可能使最低水平歪斜。现在，当1 µg物质被标定到无锈钢取样片并且允许在5cm直径的圆形上展开，它在特定的距离、角度和灯光水平下观察经常仍然是可见。5cm直径的样品面积是19.6 cm2(π x (2.5cm)2)，样品浓度是1 µg/20 cm2或 0.05 µg/cm2。这等同于1.2µg样品在25-cm2的面积上抽滤。提取1.2µg样品到溶剂中去分析结果，溶液浓度远低于1.0 µg/mL。

Table II: Spiking preparation and concentrations.

表2：标的制备和浓度

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作为清洗限度是不切合实际。用这个方法建立的VRLs将导致VRL与定量限/检测限之间的矛盾。按照法规指南，分析方法的灵敏度必须低于清洁限度，以评估清洗程序的有效性。但是如果可视限度低于分析限度，那么这个方法是不可能的。

Figure 2: Measuring 'donut' area

图2：测试环形面积

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，这个方法测试VRLs是可以接受的。但是随着人员经验的增长，VRLs起始趋势是较低的值并且分析敏感度将变成问题。任何一个选择，较多的实际定量方法是必须的，或者用这个方法生成VRLs能够被当作设备被可见污染的较多的定性和保证，在很久之前病人安全问题就对此进行过关注。

Methodology

方法学

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)

在这篇文章的第一部分对VRL描述的方法学有几个可选方案，定性和定量相结合。第二个定性选择是标定残留物在ARL和ARL低一些的水平（50%或25%），使用的标定过程如前所述。如果残留物能够被可视检测，那么就有信心能够检测到设备上的低于ARL水平的残留物，因此，目视检查也适用于检测清洁线的设备。VRL被列为“低于”检测到的最低残留物水平。对于这项研究，测定实际定量的VRLs的另外两个可选方法进行了探索。第三个方法是按照描述持续制备同样的残留物样品，通过大圆减去内部小圆的面积仅留下被覆盖的面积进行测定残留物圆环的面积，残留物如图2和图3所示用公式1：圆环面积=πr2– πr22= π(r12– r22) = π(r1– r2) (r1– r2)

Figure 3: Measuring 'donut' area.

图三：测定圆环面积

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的标定溶液结果是一个外直径5cm和内直径4.8cm的圆环的残留物，圆环残留物的面积仅是1.5 cm2，相当于直径5cm的圆，它的面积是19.6 cm2。对于1µg的残留物样品，浓度从0.05 µg/cm2变化到了0.67 µg/cm2。这将导致17 µg的抽滤样品用于25cm2的抽滤面积。

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.

第四个方法也是比较偏重定量，是去用统一的覆盖面形成残留物。因为像1 µg/ 100 µL的低标水平导致圆环残留物在较大的面积，较小残留物的面积能够通过高浓度样品小体积的标定来实现。取20 µL 75 µg/mL的标定溶液导致1.5µg残留物分布比较均一。由此产生的均匀残留物遍布在一个直径0.8cm的圆上，0.5cm2的面积和3 µg/cm2或75 µg/25 cm2浓度。

Figure 4: Measuring uniform area.

图四：测试均匀面积

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.

系列稀释制成类似的标。额外的样品在表II和图四中显示。在图四中能够看到前三个斑点分布比较均匀，而第四、五个分布不均匀。VRL能够用最低均匀分布残留进行计算，按照斑点三计算VRL是0.5 µg/cm2。虽然斑点三和斑点四是可见的，但是它们分布分布不均匀，如果试图计算VRL将导致图一测定相同的问题（例如，评估过高的面积导致VRL较低）。均匀和不均匀残留物的情况与LOQ和LOD分析测试方法相似。第三个斑点能够被定量分析，但是斑点四和五的定量是不可能尽管他们是可见的。

Discussion

讨论

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：建立可见残留限度的选择

MeOH是甲醇，ARL是可接受的清洁残留限度，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.

四个VRLs的方法每个都有自己的优势和缺点。表III对不同方法包括制备、VRL水平和主要问题进行了总结。每个方法都是合理的，只要公司规定自己的工艺过程并且清楚自己所选方法的意图和局限性。

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.

最初描述的方法在标定已知残留物数量的背景章节中，在多个取样片上减少数量，然后测量整个圆面积的干残留物，保证可见残留物能够在设备表面可以很好的检测，之前的残留物对随后的提供批次是一个安全的风险。VRL的测定在VRL水平和标定API或配方的ARL之间提供了“安全边缘”定量评估。这个方法作为人员很好的培训工具在VRL附近将获得残留物外观更好的印象，这是一个有价值的视角，特别对有其他永久标记的较老的清洗设备表面。在残留物经常出现的圆环或清洗、干燥设备的水印也有实用性。

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.

这个方法主要潜在的缺点在表1中能够看到。如果1µg标定样品是可视的，通常情况下，它将有一个0.05µg/cm2的最终浓度。擦拭样品和萃取到10mL的溶剂中将出现0.1 µg/mL的样品浓度。如果这个值在分析方法的LOQ以下，大多数分析测定技术比较接近，包括通用的清洁样品测定技术，例如高效液相色谱（HPLC）和总有机碳分析（TOC）。因为VRL被认为等同于ARL，所以希望清洗样品的测试方法有一个比残留物ARL更低的LOQ。分析测定方法可能不足够敏感，或者敏感性足够，所有东西在测定中都能检测到，那么样品面临失败的危险。

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.

潜在清洗失败的危险使用这个VRL方法是不能被论证的，因为大多数标定物、干燥的残留物面积没有被残留物占据。在低的标定浓度，形成圆环，残留物仅占据干燥圆周长附近的小部分面积。虽然这个方法测定VRLs是有效的，并且提供了通过残留物数量除以残留面积得到准确的测定，但是残留不均一导致VRL低于真实水平。VRL测定把这个方法带入歧途，但是如果企图把它和分析方法的LOQ相关联。不管怎样，用这个方法建立的VRLs与ADE计算出的清洁限度比较确认可视性检查适用于清洗确认；同时测定可视限度和清洗限度之间的“安全边界”有多宽。

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.

之前描述的第二种方法最简单，是直接标定取样片在ARL更低的水平（例如50%）。如果标定的残留物是可视的，为可视残留物在设备表面被检测提供保证，以前的残留物成为了随后提供批次的一个安全风险。这个方法避免了低水平的实际问题并且也有速度的优势，样品制备比较迅速并且不需要计算。每个残留使用的取样片较少，因此，多个残留物能够同时定性。如果公司希望建立高效时间结构或迅速测定的VRL程序用多个距离和视角检测可视残留物的限制，这个方法可以推荐。这个方法也用于培训或很多人的资格确认，比如所有设备清洗人员和检测人员。这种方法也实用于残留物最常表现为环状或清洁干燥设备上的水印。

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.

这个方法的缺点是它仅提供了定性测试，而不能确定VRL和ARL之间的“安全边缘”。次要的缺点是如果被标定残留物不是可视的，不能确定ARL和被标定的水平之间的水平可能仍然是可见的，额外的取样片不得不标定直到观察到可视残留物。对于纯定性的方法最大的缺点是如果残留物是可视的且水平低于计算出的清洗ARL那么可视限度应该是实际上的清洗ARL。但是，如果一个公司声明他们在战略上看看VRLs，仅从定性角度和较低的标定水平提供置信水平去确认被可视残留物污染的设备不能认为是清洁的，用计算出的ARLs作为他们的清洁限度，这应该是可行的方法和站得住脚的中介。

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.

第三个方法是用与前两个方法相同的标定技术，但是仅测量被残留物覆盖的圆环面积。这个方法有几个优势，取样片的标定是简单的，残留物出现在清洁干燥的设备上，并且最为重要的是这个方法导致VRL更真实的检测，因为它仅包括了残留物覆盖的面积。更高的VRL测试也应该在分析测定方法的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.

这个方法最显著的缺点是圆环厚度潜在的不一致和圆环厚度后续的主观测量。圆环的内边可能不能很好的定义和进行主观的评价来测量圆环的内边缘。圆环越宽，干燥的残留物浓度结果就越低。在使用这个方法生成官方VRL数据之前，这个问题需要在协议中处理。

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.

第四种方法的目的是为了有均匀的残留相对于圆环类型的残留。这个方法的优势包括更真实的VRL仅基于残留物覆盖的面积，而且事实上，残留越小占据的空间越小因此取样片更少，生成较多的残留物VRLs有较少的取样片。

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.

这个方法潜在的缺点包括：精确吸取较少的体积（20 µL）和可见残留物的LOQ和LOD的概念。在某一时刻，残留物仍然是可见的，但是浓度不能被精确测定。

结论

不同方法建立的VRLs被提出和讨论。四个方法中的每一个都是可行的，只要讲清楚整个清洗验证的主计划和持续性应用。两个更精确的定量方法被定义，它们看上去大致相当于VRLs，但是每个的实用性只能通过生成的数据和并列的对比来评估出更好的。