蒸汽灭菌的常见错误

Most mistakes regarding the programming and operation of typical steam sterilizers are related to the basic principles of steam sterilization.

常见蒸汽灭菌柜的大多数程序和操作错误都与蒸汽灭菌的基本原理有关。

 

1. Containers with closed valves, empty glass bottles with tightened screw caps or secured aluminum foil are placed in the sterilizer.

灭菌柜中放入关闭阀门的容器、旋紧瓶盖或紧扣铝盖的空玻璃瓶。

 

As a result, steam cannot directly contact the inside surfaces and sterilization does not occur. This problem can be resolved by assuring that all items in the sterilizer have a way for the steam to get in and the air to get out. If there is uncertainty about whether an item’s configuration, set-up, packaging, or orientation will allow adequate steam penetration, a thermocouple, chemical and/or biological indicator can be placed inside the item to be certain.

这样， 蒸汽就不能直接接触到内表面，不能进行灭菌。 这一问题可通过确保灭菌柜中的物品有蒸汽进入和空气排出的通道来解决。 如果不确定物品的配置、设置、包装或方位是否允许充分的蒸汽穿透，那么可以通过在物品内部放置热电偶、化学和/或生物指示剂来确定。

 

2. Pouched and/or heavily wrapped items are tightly packed in the chamber.

袋装和/或严密包裹的物品紧实地装载在腔室内。

 

As a result, air may remain trapped in the items after the preconditioning phase and prevent sterilization. Items should not be overwrapped, and sufficient space should be maintained between load items. The preconditioning vacuum and pressure pulses must be set correctly to attain complete air removal from the load. Typically, four (or more) preconditioning vacuum pulses should be programmed to reach at least 28 in (711 mm) Hg vacuum ((1.0 psia or 6.9 kPa (absolute)) to assure sufficient air removal for worst case loads. Some very dense loads may require a short (2 to 5 min) hold phase at peak preconditioning vacuum to allow time for trapped air to be removed. Preconditioning pressure pulses should be programmed for 3 to 5 psig ((21 to 34.5 kPa (gauge)). Higher pressures set for prevacuum pressure pulses can result in an excessive amount of superheat and difficulties with temperature stabilization during the first few minutes of the exposure phase.

这样， 预调节阶段后空气可能仍然滞留在物品中，阻碍了灭菌。 物品不应过度包裹，且应在物品间留有足够的空间。 预调节真空和压力脉动必须正确地设定，以完全排除装载内的空气。 通常， 应该程序设定达到 28 in (711 mm)汞柱((1.0 psia or 6.9kPa (绝对压力))真空且四次(及以上)预真空脉动，以确保最差装载情况下空气排除充分。 有些非常密集的装载在预调节真空峰值时可能需要一段较短（ 2-5min）的保持时间，从而排除滞留的空气。 预调节真空脉动应设定为 3~5 psig ((21~34.5 kPa (表压))。 预调节压力脉动可能造成暴露阶段前几分钟时的过热和温度稳定困难。

 

3. Heavier items are placed on top shelves.

较重的物品置于货架顶层。

Water droplets and/or stains are observed on the outside of wrappers of items placed on the mid to lower shelves after the sterilization cycle is complete. Because the items are not dry, they cannot be aseptically removed from the sterilizer. Condensation is the natural result of steam contact with the cooler surfaces of the load. The condensate will fall from shelf to shelf. The denser the load item, the more condensate is created. Therefore, place heavier items on the bottom shelf. In addition, consider placing a cotton sheet or lint free towels on each sterilizer loading cart shelf prior to loading to allow the condensate to be absorbed. This also aids in drying. As the condensate wicks into the sheet or lint free towels, the condensate surface area is greatly increased and evaporates much more rapidly during the drying phase than the same amount of condensate in a droplet or a puddle.

灭菌循环结束后， 置于中间和低层的物品包装外表面可以看到水滴和/或污迹。 由于物品不是干燥的，不能从灭菌柜中无菌地取出。 冷凝是蒸汽接触物品冷表面时的自然结果。冷凝水会从货架上滴下。物品的放置越密， 产生的冷凝水越多。 因此， 将更重的物品放置于低层。 另外，在装载前考虑在各灭菌装载货架上放置纯棉布或无绒毛巾来吸收冷凝水。这也有助于干燥。由于冷凝水浸入棉布或无绒毛巾，冷凝水表面积大大增加， 与同量冷凝水滴相比干燥阶段的气化速率也大大提高。

 

4. Load is too dense or items are positioned incorrectly in the load.

装载过于密集或物品在装载中摆放位置不对。

 

As a result, wet or damp items are observed at the end of the cycle. Wrapped items positioned so that condensate is allowed to collect will not be dried. Items should be positioned so that the condensate is allowed to flow downward. Items (wrappers, pouches, filters, or other porous biological barriers) that remain wet at the end of cycle cannot prevent contamination of the load when removed from the sterilizer. As the load cools outside the sterilizer, the water in the wrapper will be drawn into the wrapped item. Any contamination that is present in the environment can be drawn through the sterile barrier along with the water. There are numerous other possible causes for wet loads.The most common are:

因此， 循环结束时发现物品变得潮湿。 放于会收集冷凝水的位置的包裹物品不会被干燥。 物品的放置应允许冷凝水向下流动。 物品（包裹、 呼吸袋、过滤器或其它多孔生物滤膜） 由于在循环结束时仍是湿的，在从灭菌柜取出时不能避免装载的污染。由于在灭菌柜外冷却，包裹上的水会浸入到被包裹的物品中。 任何环境中的污染源都可能随水一起穿过无菌屏障。 还有许多其它原因可能会造成装载潮湿，最常见的是：

 

a. Insufficient drying vacuum level or time programmed

程序设计干燥真空水平或时间不够

 

b. Rubber or plastic items in pouches (i.e., rubber stoppers, plastic tubing) may require additional drying (a pulsed air or heated pulsed-air drying process is recommended for these items)

袋装的橡胶或塑料制品（如橡胶塞、塑料管）可能需要额外的干燥（ 对于这些物品，推荐采用脉动空气或脉动热空气干燥工艺）

 

c. Wet steam

湿蒸汽

While there is no single solution to eliminating wet loads, it’s likely that experimenting with drying time, repositioning items, reducing load density, modifying cycle settings, and investigating steam quality will resolve the problem.

由于没有哪个方案一定能有效消除装载潮湿，很可能通过试验干燥时间、重新放置物品、降低装载密度、改进循环设置和调查蒸汽质量可以解决这个问题。

 

5. Pouches are placed flat on the sterilizer shelves or stacked on top of one another.

呼吸袋平放于灭菌柜货架上或叠放在一起。

 

As a result, pouches may have water droplets inside and cannot be aseptically removed from the sterilizer. Typical cause is when the condensate naturally created when steam penetrates the pouch and contacts the surface of the item within is not removed during the post-conditioning drying phase. Pouches should be spaced properly and placed in rack that holds the pouch on its edge (as figure) to prevent pooling of the condensate inside the pouch. Pouches should not be placed flat on the sterilizer shelf. Pouches should not be overloaded. Remember that more mass means more condensate.

这样， 呼吸袋可能会有水滴在内部， 不能从灭菌柜中无菌地取出。 通常原因是蒸汽穿透呼吸袋和接触物品表面时会自然形成冷凝水，这些冷凝水在后调节干燥阶段没能除去。 呼吸袋应留有足够的空隙，并放置于搁架上（如图），以防止呼吸袋内部冷凝水聚集。 呼吸袋不应平放于灭菌架上。 呼吸袋不应叠放。 记住， 物品越多，冷凝水越多。

Sufficient drying vacuum level and time should be programmed to allow for complete evaporation of the condensate. Wet steam should be corrected. Double pouching may require additional prevacuum pulses with dwell time at maximum vacuum and increased drying time. Doubled pouches should never be assembled so that the items inside cannot be seen.

Pouch flaps should not be folded over.

程序应设定足够的干燥真空水平和时间， 使冷凝水完全气化。 湿蒸汽应调节为饱和蒸汽。 双层呼吸袋可能需要额外的预真空脉动， 在最大真空时保持一定时间并增加干燥时间。 双层呼吸袋绝不应以看不到内部物品的方式组装。 袋口不应折叠起来。

 

6. Liquids in vented containers are placed in a deep pan to catch boil-over (slow exhaust cycle).

通气容器中的液体放置于深托盘内，以收集沸溢的液体（在缓慢排气循环）。

 

The pan will hold water and it will hold air. The steam cannot contact the surfaces within the pan because of the trapped air, and they will not be sterilized. The solution is to eliminate the pan and adjust the sterilizer slow exhaust rate to prevent boil-over. A shallow pan, less than 1” (25 mm) deep, can be used in the event that a small amount of boil-over cannot be eliminated

by adjusting the slow exhaust rate.

托盘会容纳水也会滞留空气。 蒸汽不能在托盘内受滞留空气的阻碍而不能接触到表面，因此达不到灭菌效果。 解决方法是去除托盘，调事灭菌柜的缓慢排气速率，防止沸溢。 浅托盘，少于 1” (25 mm)深， 可用于少量沸溢不可避免时的情况。

 

7. “Overcooked” Media

培养基“加热过久”

 

Over sterilization of media will caramelize the sugars and render the media useless. The typical overkill approach is not recommended for sterilization of media. The exposure phase should be programmed to achieve the desired SAL and no longer. Use of a load probe and F0 exposure control is recommended for sterilization of media in containers larger than 100 ml (3.4 oz). As illustrated in Figure 8, F0 is a calculation of the equivalent exposure at temperatures other than 121.1°C (250°F). As the liquid is heated, the calculated F0 (from the load probe temperature) is accumulated until the selected F0 exposure value (minutes) is achieved, at which point the cycle proceeds to the exhaust/cooling phase. For example, on the graph, the kill rate on the same population of organisms is half as effective at 118°C (245°F) as at 121°C (250°F). Therefore, at 118°C (245°F), it will require twice the exposure time to kill the same number organisms.

培养基过度灭菌会使糖分焦化，使培养基失效。培养基灭菌不推荐采用典型的过度杀灭方法。 程序应设计暴露阶段， 以恰好达到所需的 SAL。 在大于100 ml (3.4 oz)的容器内的培养基推荐采用装载探头和 F0 值暴露控制灭菌。 如图 8 所示， F0 是 121.1°C(250°F)以外的温度下计算等效暴露。 随着液体被加热， 计算出的 F0（从装载探头温度） 逐渐累积，直到达到所选的 F0 暴露值（分钟）， 此时循环进行至排气/冷却阶段。例如，从图上可以看出， 在 118°C(245°F)时同样数量微生物的杀死率是在 121°C(250°F)时的一半。 因此，在 118°C (245°F)时，需要两倍的暴露时间才能杀死同样数量的微生物。

Figure 8. F0 as a function of temperature.

图 8. 温度函数 F0

A common formula for calculating the F0 value is:

计算 F0 值的常见公式是：

where:

其中，

· L is lethal rate of bacterial spores

· L 是细菌孢子致死率

· t is exposure time, [s]

· t 是爆热时间， [s]

· T is exposure temperature, [°C]

· T 是爆热温度， [°C]

· z is a constant, [°C]

· z 是常数， [°C]

The constant z describes the slope of the thermal death curve. The widely accepted value for z is 10°C (18°F) in steam sterilization.

常数 z 描述热力致死曲线的斜率。 对于蒸汽灭菌，广泛认同的 z 值是 10°C (18°F)。

 

8. Using cold water for vacuum pump that is too hot.

真空泵使用的冷却水过热

 

As a result, the vacuum pump may not be able to reach 1.0 psia (6.9 kPa). The heart of the prevacuum sterilizer is the water-ring vacuum pump. The efficiency and maximum vacuum capability of a water-ring vacuum pump are adversely affected by higher water temperatures typically encountered during the summer months. During operation, the water within the pump is heated by mechanical friction and heat energy from the sterilizer chamber. If the temperature of the water inside the pump reaches 39°C (102°F) during the preconditioning or post conditioning vacuum peak, the water inside the pump will boil at ≤ 1.0 psia (6.9 kPa) and cause cavitation. In this case, the recommended preconditioning vacuum level of 1.0 psia (6.9 kPa)

cannot be achieved in the sterilizer chamber. A common “work-around” for this situation is to change the set point of the prevacuum pulses to a level that can be achieved. Insufficient air removal can be the result unless the number of vacuum pulses is increased, causing longer cycle times and less effective air removal. Internal pump temperatures higher than 39°C (102°F) are often observed during the summer months if the water supplied to the pump is not cooled. Chilled water is ideal, but typically too expensive to use in a sterilizer vacuum pump arrangement in which the water flows from the vacuum pump to drain. The recommended solution is a recirculation/cooling system for the vacuum pump water that uses chilled water in a closed loop heat exchanger. This configuration is eco-friendly as it saves a significant amount of water. In addition, the vacuum pump efficiency is not subject to seasonal water temperature fluctuations.

这样，真空泵可能达不到 1.0 psia (6.9 kPa)。真空灭菌的核心是水环真空泵。在夏季期间经常发生水温过高，这可能会对水环真空泵的效能和极限真空度造成不良影响。在操作期间，泵内的水由于机械摩擦和灭菌腔室的热量被加热。如果预调节或后调节真空期间泵内的水温达到 39°C (102°F)，泵内的水在≤ 1.0 psia (6.9 kPa)真空下会沸腾，造成气蚀。这种情况下，灭菌腔室内就达不到推荐的真空水平1.0 psia (6.9 kPa)。这种情况常见的“应对措施”就是改变预真空脉动的真空设定值，降为可达到的水平。这可能造成排气不足， 除非增加真空脉动次数，从而造成循环周期延长，排气效率下降。 如果真空泵进水未经冷却，夏季时经常发现泵内温度高39°C(102°F)。 冷冻水是理想选择，但对于灭菌柜真空泵来说成本过高，因为真空泵的出水直接排放。 推荐措施是真空泵配循环冷却水系统，采用冷冻水和闭路循环换热器。 这种配置可节省大量的水， 经济环保。此外，真空泵效率不受水温季节性波动的影响。

 

9. Load probe is available, but not used.

有装载探头但没有使用。

Most modern sterilizers include (optional) an RTD load probe and F0 exposure control for use in liquids sterilization, but many times the probe is not used. If equipped with a load probe, the exposure can be controlled by the temperature of the liquid rather than the temperature in the drain line. Without the load probe, the temperature of the liquid is not known and can only be estimated, resulting in inadequate (non-sterile) or excessive F0 (overcooked). The load probe should be placed in a container of water approximating the volume of the largest volume of liquid being sterilized. Load probe control/F0 must then be selected in the sterilizer control settings.

多数现代灭菌柜配备（可选）了一个 RTD 装载探头和 F0 暴露控制，用于液体灭菌的控制，但是许多情况下这一探头都没有被使用。 如果配备了装载探头， 暴露可以依据液体温度来控制，而不是排放管路内的温度。 如果没有装载探头， 液体温度是未知的，只能估算， 因此造成灭菌不充分（非无菌）或过度（ 加热过久）。装载探头应置于含水容器内，容器内盛水体积约为待灭菌液体的最大体积。 装载探头控制/F0必须在灭菌柜控制设置中选择。

 

10. Pressure/vacuum rate control is available, but not used.

卸压/抽真空速率控制可用，但没有使用。

 

Most modern sterilizers include (optional) rate control for the vacuum and pressure ramps, but many times the rate control is not used. When no pressure rate control is applied steam will enter the chamber at maximum velocity during the preconditioning pressure pulses, which creates a superheat problem and EN285 compliance problems as discussed earlier. Slowing the pressure rate allows time for superheat to dissipate during the ramp up.

多数现代灭菌柜具备（可选） 抽真空和卸压速率的控制功能，但许多情况下该速率控制并没有被使用。 如果没有采用卸压速率控制，在预调节卸压阶段，蒸汽将以最大速率进入腔室，从而造成过热的问题和 EN285 符合性问题。 减缓卸压速率使卸压过程中的过热有时间分散。

 

When no vacuum rate control is applied the chamber will depressurize at the maximum rate of the vacuum pump. The typical problem associated with this is burst pouches. Slowing the vacuum rate allows time for the pouch internal pressure to equilibrate and prevents bursting during the preconditioning and post conditioning vacuum phases.

如果没有采用抽真空速率控制， 腔室将以真空泵的最大速率形成真空。这造成的常见问题是爆袋。在预调节和后调节抽真空阶段， 减缓抽真空速率使呼吸袋内部压力有时间平衡，并防止爆袋。

 

others: 

其他注意事项:

Steam sterilizers typically have a steam jacket to insulate the chamber and improve temperature distribution. When a steam jacket is present, it is ordinarily operated at lower steam pressure and temperature than the chamber to avoid superheat.

蒸汽灭菌柜通常有一个蒸汽夹套来隔离腔体并提高温度分布。为避免过热，夹套的蒸汽操作压力和温度通常比腔体要低。

 

Pipe slopes should be adequate to promote drainage and condensate removal

管路有一定的坡度以利于污水和冷凝水的排放。

 

Welds should meet applicable materials and joining standards

焊接应满足适用的材料以及焊接标准。

 

If the incoming steam pressure is above the manufacturer's recommendations, additional valves (e.g., modulating steam, pressure reducing, pressure relief, shut-off) should he considered.

如果进口蒸汽压力超出供应商的建议值，应当考虑使用其他阀门（如蒸汽调节阀，减压阀，泄压阀，截流阀）。

 

An appropriate thermostatic steam trap combined with floating ball should be used to quickly remove large quantities of air and/or condensate from the chamber.

为了快速移除腔体中大量空气和/或冷凝水，应当选用一个合适的恒温式疏水阀以及浮球。

 

公众号

GMP办公室

• 专业的GMP合规性研究组织

• 国内外（FDA、EMA、MHRA、CFDA、WHO、PIC/S等）GMP法规解读；

• 国内外制药行业GMP监管动态；

• GMP技术指南（ISPE、PDA、ISO、ASTM等）分享