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Lyophillizer Validations, Aseptic Process simulations design-Lyo process and Rationale for Hold duration of Partially Stoppered Media Filled Vials-In compliance with Revised EU Annex-1 2022 冻干机验证，无菌工艺模拟设计-冻干工艺和部分密闭培养基灌装小瓶保持时间的基本原理-符合修订后的欧盟附录1 2022 2024.02 Part IAfter two draft documents from 2017 and 2020, more than 6,000 comments and much speculation about the final publication date, it is finally here: Yes, the long-awaited final version of the new 58 pages of EU Annex 1 'Manufacture of Sterile Medicinal Products' was published on August 25, 2022. The deadline for entry into force was August 25, 2023. 经过2017年和2020年的两份草案文件、6000多条评论以及对最终发布日期的众多猜测，它终于来了：是的，期待已久的欧盟附录1“无菌药品生产”新58页最终版本于2022年8月25日发布。生效截止日期为2023年8月25日。 The document has increased in size by another six pages from the draft version, but its basic structure into eleven areas has remained the same. Among these, sections 8.121 to 8.126 on freeze-drying and product transfer were covered in this article and the deadline for entry into force is two years and will thus become mandatory on August 25, 2024. 该文件比草案版增加了六页，但其基本结构保持不变，分为十一个领域。其中，第8.121至8.126节关于冻干和产品转移的内容涵盖在内，生效期限为两年，因此将于2024年8月25日强制执行。 The scope of Annex 1 applies to all sterile medicinal products manufactured in the European Union and the UK, as well as those manufactured elsewhere and exported into the European Union. 附录1的范围适用于在欧盟和英国生产的所有无菌药品，以及在其他地方生产并出口到欧盟的所有无菌药品。 As we all know, the pharmaceutical industry is facing the new challenges of integrating the new EU GMP Annex 1 requirements into its current and new facilities. 众所周知，制药行业正面临着将新的欧盟 GMP 附录1要求整合到其现有和新设施中的新挑战。 The purpose of this white paper is to introduce Annex 1 and to describe the significant changes and the scientific/regulatory reasons behind the design of aseptic process simulation for lyophilized drug products with special considerations in key validation aspects of lyophillizer. 本白皮书的目的是介绍附录1，并描述冻干药品无菌工艺模拟设计背后的重大变化和科学/监管原因，并特别考虑冻干机的关键验证方面。 Introduction 介绍The “Media Fill,” or “Broth Fill,” technique, is one in which a liquid microbiological nutrient growth medium is prepared and filled in a simulation of a normal manufacturing operation. The nutrient medium processed and handled in a manner which simulates the “normal” manufacturing process as closely as possible with the same exposure to contamination (from operators, environment, equipment, and surfaces) as may occur during routine manufacture. The sealed containers of medium thus produced are then incubated under prescribed conditions and examined for evidence of microbial growth, and thus can obtain indication of the level of contaminated units produced. “培养基灌装”或“肉汤灌装”技术是一种模拟正常生产操作制备和灌装液体微生物营养生长培养基的技术。营养培养基的加工和处理方式尽可能模拟“正常”生产工艺，并尽可能暴露于与日常生产工艺中相同的污染（来自操作员、环境、设备和表面）。然后将这样生产的培养基密封容器在规定条件下进行培养，并检查是否有微生物生长的证据，从而可以指示生产的污染单元的水平。 Purpose of Aseptic Process simulation 无菌工艺模拟的目的The goal of a media fill is to demonstrate that the manufacturer can follow the routine aseptic production process using sterile media without contamination (evaluate and justify the aseptic capabilities of the process, the people and the system). Hence in this article, we ensure lyo media fill should primarily validate the filling, transportation and loading and unloading aseptic operations with certain duration of filled vials in the lyophillizers. 培养基灌装的目的是证明生产商能够使用无菌培养基进行日常无菌生产工艺而不会受到污染（评估并证明流程、人员和系统的无菌能力）。因此，在本文中，我们确保冻干培养基灌装应主要验证在冻干机中灌装、运输和装卸灌装小瓶的无菌操作，并保持一定时间。 During aseptic process simulation for lyophilized product, filled vials are aseptically filled in the normal way (similar to liquid filling), but the closures (which are of a single slotted or double slotted) are not fully inserted in the filled vials and transported aseptically through conveyor/ALUS/robotics to the lyophilizer shelves under Grade A atmosphere. 在冻干产品的无菌工艺模拟中，已灌装的小瓶以正常方式进行无菌灌装（类似于液体灌装），但密封（单槽或双槽）未完全插入已灌装的小瓶中，而是通过传送带/ALUS/机器人无菌运输到A级大气下的冻干机架子上。 Loading of media filled vials in the lyophilizer 将装有培养基的小瓶装入冻干机Shelf to be pre-cooled to 20-25°C. The media filled vials are loaded in the sterilized lyophilizer (using suitable in-house designs such as automatic loading/Manual loading using fences). At the completion of the loading process, it is important to ensure all filled, partially stoppered vials are loaded into the lyophilization chamber. 搁板预冷至20-25°C。将装有培养基的小瓶装入灭菌冻干机（使用合适的内部设计，如自动装载/使用围栏手动装载）。装载工艺完成后，重要的是确保所有已装满、部分封闭的小瓶都装入冻干室。 Product lyophilization process 产品冻干工艺Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. 冻干或冻干是将产品冷冻并置于真空条件下，除去产品中的水分的工艺，使冰直接从固态变为蒸汽，而无需经过液态。 Entire Lyophillizer needs to be simulated by loading? 整个冻干机都需要通过装载进行模拟吗？Instead of filling and loading all the shelves with media filled vials, covering top, middle and bottom shelves can be preferred. These shelves shall be loaded with filled partially stoppered vials to mimic the routine process. The shelf numbers 1, 3, 6, and 10 shall be selected to cover the loading activity (top to bottom loading) if the design of lyophilizer is with 10 shelves and this process simulation will cover all the shelves are loaded into the freeze dryer to cover the whole freeze-drying chamber and process. 可以优先覆盖顶部、中部和底部搁板，而不是用装满培养基的小瓶填充和装载所有搁板。这些搁板应装载已填满且部分封闭的小瓶，以模拟日常工艺。如果冻干机的设计有10个搁板，则应选择搁板编号1、3、6 和 10来覆盖装载活动（从顶部到底部装载），并且此工艺模拟将覆盖装入冻干机的所有搁板，以覆盖整个冻干室和工艺。 What will happen if it is simulated for longer hours of holding similar to product lyophilization? 如果模拟类似于产品冻干的更长时间的保存，会发生什么情况？Exact simulation of media fill as that of product lyophilization will have possible reduction of microbiological levels after aseptic manipulation which will not solve the purpose of aseptic process simulation. 与产品冻干一样的培养基灌装的精确模拟，可能会降低无菌操作后的微生物水平，但这并不能解决无菌工艺模拟的目的。 Carrying out a lyophilization cycle and freezing the media will be same simulation as that of product lyophilization, however this freezing of media will reduce microbial levels of some contaminants. 执行冻干循环，并冷冻培养基，将与产品冻干的模拟一致，但是这种培养基的冷冻会降低某些污染物的微生物水平。 In aseptic process simulation of lyo process the scope is not to check the lethality of freezing and its effect on microorganisms that might be present. Hence freezing of media and the formation of ice crystals is unfavorable to microorganisms hence this should be avoided. 在冻干工艺的无菌工艺模拟中，范围不在于检查冷冻的致死率及其对可能存在的微生物的影响。因此，培养基的冷冻和冰晶的形成对微生物是不利的，因此应避免这种情况。 Unfrozen media and complete vacuum 解冻培养基并完全真空If media is not frozen, then the lyophilizer may get contaminated due to unfrozen media which is left under vacuum similar to the product lyophilization vacuum level. If complete vacuum is drawn then it may cause the media solution to get out from the containers and contaminate the lyophilizer as well fluid loss from the containers and finally this may have serious effect on the viability of the microorganism and the ability of the media to support microbial growth will be impacted this will in turn make aseptic process simulation invalid. 如果培养基未冷冻，冻干机可能会因未冷冻的培养基处于与产品冻干真空度相似的真空环境中而受到污染。如果完全抽真空，则可能导致培养基溶液从容器中流出并污染冻干机，以及容器中的液体损失，最终可能对微生物的生存能力产生严重的影响，培养基支持微生物生长的能力将受到影响，这反过来会使无菌工艺模拟无效。 Hence a complete vacuum as specified for the lyophilization process should not be drawn during the media fill. 因此，在培养基灌装过程中，不应抽取冻干工艺中规定的完全真空。 Hold time duration in the lyophillzer 在冻干机中的保持时间In general, the course and processing time should be long enough to challenge or stress the process, interventions, the supporting environment, and the operators. All efforts should be made to perform all routine interventions. The filled partially stoppered vials will remain inside the lyophilizer chamber for minimum 12 hours (Author’s experience for hold time duration). 一般而言，工艺和处理时间应足够长，以挑战或强调工艺、干预措施、支持环境和操作人员。应尽一切努力执行所有日常干预措施。已灌装的部分封闭的小瓶将在冻干机室内保留至少12小时（作者对该保留时间长度的经验）。 The duration of the media filling stated above represents lyophilization cycle for one shift. 上述培养基灌装的持续时间代表一个班次的冻干周期。 The integrity of the lyophilizer chamber is tested as a part of preparation process (ie) during post sterilization cycle. Therefore, no additional benefit is drawn by holding the media filled vials for longer duration (such as product lyophillization cycle), as the lyophilizer’s integrity is maintained throughout this cycle. Hence, it is not necessary to carry out lyophilization cycle as per the actual drug product. 冻干机腔体的完整性是制备工艺的一部分，即在灭菌后循环期间进行测试。因此，将装有培养基的小瓶放置更长时间（例如产品冻干循环）不会带来额外好处，因为冻干机的完整性在整个循环工艺中都得到保持。因此，没有必要根据实际药品进行冻干循环。 Secondly, as compared to lyophilization hold duration for filled vials in lyophillzer the chances of contamination for partial stoppered vials is more during the process stages such as empty vial transportation, media fillings and transportations, interventions, samplings, half stoppering, loading of vials into lyophillizer, etc. 其次，与冻干机中已灌装小瓶的冻干保持时间相比，在空瓶运输、培养基灌装和运输、干预、取样、半封塞、将小瓶装入冻干机等过程阶段，部分封塞的小瓶受到污染的可能性更大。 So how long the APS process shall be simulated? 那么APS过程要模拟多久呢？Typically 600 mbar to 700mbar, few manufacturers used maximum of 900 mbar of partial vacuum and held for about 12 hours and few manufacturers held it for two to three hours and then vacuum in chamber gets broken by sterile filtered compressed air (used to back fill the chamber, instead of the nitrogen) and full stoppering of vials shall be performed using the stoppering mechanism at ambient pressure and open the door. 通常为 600 mbar 至 700mbar，少数生产商使用最大900mbar的部分真空并保持约12小时，少数生产商将其保持两至三个小时，然后用无菌过滤的压缩空气（用于回填腔室，而不是氮气）打破腔室内的真空，并且应在环境压力下使用封塞机构对小瓶进行完全封塞并打开门。 Finally unload the vials aseptically and transfer to sealing machine. All these activities shall be performed in class 100 (Grade A). 最后以无菌方式卸下小瓶并转移到封口机。所有这些活动应在100级（A级）下进行。 Possible questions from regulatory agencies for Lyo media fills 监管机构可能对培养基灌装提出的问题ŸIs the aseptic handling of lyophilized products validated by media fills? 冻干产品的无菌处理是否通过培养基灌装进行验证？ ŸIn the aseptic process, is lyophilization simulation performed during media fill? 在无菌工艺中，培养基灌装时是否进行冻干模拟？ ŸIs the maximum amount of time the vials are held prior to lyophilization simulated during media fills. If vials are not sealed in lyophilization chamber, is the maximum hold time prior to stoppering simulated in media fills? 培养基灌装工艺中是否模拟了冻干前小瓶保持的最大时间。如果小瓶未在冻干室中密封，培养基灌装工艺中是否模拟了封塞前的最大保持时间？ ŸDuring validation, what level of vacuum is pulled on the lyophilization chamber? 验证期间，冻干室的真空度是多少？ ŸHow long do media fill vials remain in the lyophilization chamber under vacuum? How does this compare to commercial lots? 培养基灌装瓶在真空条件下可在冻干室中保存多长时间？与商业批次相比如何？ ŸDoes the process simulation result in freezing of the media? Note that this process simulation should not include freezing of the media. 工艺模拟是否会导致培养基冻结？请注意，此工艺模拟不应包括培养基的冻结。 ŸIs environmental monitoring performed during loading of the lyophilizer both during production and as well as during validation? 在生产和验证过程中，冻干机的装载过程中是否进行环境监测？ ŸDoes the firm have data on growth promotion of the media? Are growth promotion tests done on vials after incubation is completed? 公司是否有关于培养基促生长的数据？培养完成后，是否对小瓶进行促生长测试？ ŸIs environmental monitoring performed during unloading of the chamber during production as well as during media fill validation? 在生产过程中卸载腔室时以及培养基灌装验证期间是否进行环境监测？ ŸWhat is used to break the vacuum during media fills (nitrogen, air, other gas)? 在培养基灌装期间用什么来破坏真空（氮气、空气或其他气体）？ In my view, with practical experiences in conducting and designing several aseptic process simulation programs - would recommend few key rationales for bracketing concepts: 在我看来，凭借在执行和设计多个无菌工艺模拟程序方面的实践经验，将为括号法概念推荐几个关键理由： Vial and Stopper Size 小瓶和瓶塞尺寸The neck size of the vial is considered as a deciding parameter for the media fill and other parameters like body diameter are considered as non-deciding parameters for the media fill. The vials having neck diameters of 13 mm, 20 mm and 32 mm are generally used in industry. The size of the vial shall be considered while deciding the worst case for the media fill. 瓶颈尺寸被视为培养基灌装的决定性参数，而瓶身直径等其他参数则被视为培养基灌装的非决定性参数。行业常用瓶颈直径为13毫米、20毫米和32毫米的小瓶。在决定培养基灌装的最差条件时，应考虑小瓶尺寸。 The size of the rubber stopper is deciding parameter of the media fill and hence is considered as a critical parameter. The rubber stoppers of size 13 mm, 20 mm and 32 mm are generally used in industry shall be considered while deciding the worst cases for the media fill. 橡胶塞的尺寸是培养基灌装的决定性参数，因此被视为关键参数。行业常用的13毫米、20毫米和32毫米尺寸的橡胶塞在确定培养基灌装的最差条件时应予以考虑。 Type of Vial and Stoppers 瓶子和瓶塞的类型The tubular or molded vials are in use. For the media fill the tubular vials are considered as worst case since the tubular vials are having lesser weight and have larger movement or tendency to toppl. 管状或模制小瓶正在使用中。对于培养基灌装，管制小瓶被认为是最坏的情况，因为管制小瓶重量较轻，且移动或倾倒倾向较大。 The stoppers of the Bromo butyl and chlorobutyl are generally used and the type of the stopper is not a deciding parameter for the media fill and stopper of any type can be used for the media fill. 一般使用溴丁基和氯丁基瓶塞，瓶塞类型不是培养基灌装的决定性参数，任何类型的瓶塞均可用于培养基灌装。 Source of Vial 小瓶来源The source of vials is not a deciding parameter for the media fill and hence any source of vials can be used. 小瓶的来源不是培养基灌装的决定性参数，因此可以使用任何来源的小瓶。 Finishing and color of Vials and Stoppers 小瓶和瓶塞的表面处理和颜色The finish of vial viz: Blow back or non-blow back and treated or non-treated does not play a deciding role in media fill and hence the vial of any finish can be used. The clear glass vials shall be used for the media fill to facilitate the visual inspection of the vials. 小瓶表面处理，即：回吹或非回吹、处理或未处理，在培养基灌装中不起决定作用，因此可以使用任何表面处理的小瓶。应使用透明玻璃小瓶进行培养基灌装，以方便对小瓶进行目视检查。 Design of stopper 瓶塞设计The rubber stoppers coated and noncoated are used generally but not as deciding parameter for the media fill. But design of stoppers are important and plays a deciding factors e.g. 2-leg, 3-leg igloo or flat-teflon, serum etc 涂层和无涂层橡胶塞通常被使用，但不是培养基灌装的决定性参数。但塞子的设计很重要，起着决定性作用，例如2脚、3脚圆顶或扁平聚四氟乙烯、血清等 Filling Speed 灌装速度Slowest line speed with the highest opening diameter, i.e long time the unit is open and a wide area, where contamination could fall into the unit. Also, usage of slow line speed is generally appropriate for evaluating manufacturing processes with prolonged exposure of the sterile drug product and containers/closures in the aseptic area. 最慢的线速和最大的开口直径，即单元长时间敞口且面积较大，污染物可能落入单元中。此外，使用慢线速通常适用于评估在无菌区域中长时间暴露无菌药品和容器/封盖的生产工艺。 Conduct each simulation study run with a single line speed by simulating the worst-case condition. E.g.: using the slowest fill speed for the biggest container size and fastest speed for the smallest container size. 通过模拟最差条件，以单一线速运行每次模拟研究。例如：对最大容器尺寸使用最慢的灌装速度，对最小容器尺寸使用最快的灌装速度。 Fill Volume 灌装体积The volume of medium must be sufficient to provide contact with all container closure seal surface on inversion and visibility for the detection of microbial growth. The quantity of sterile SCDM medium shall be considered for filling in the vial with standard different sizes are as follows: 培养基的量必须足以在倒置时与所有容器封盖密封表面接触，并能检测微生物生长。应考虑无菌SCDM培养基的数量，以灌装标准不同尺寸的小瓶，如下所示：
Pulling and Releasing of Partial Vacuum 抽取和释放部分真空Pull the condenser of lyo to -40 °C and start the vacuum pumps. The loaded vials are exposed to the partial vacuum cycle. 将冻干器的冷凝器拉至 -40 °C 并启动真空泵。已装载的样品瓶暴露于部分真空循环中。 First Pull of Vacuum 第一次抽真空Pull the vacuum to 500-700[1] mbar, after achieving the vacuum of 600 mbar, and maintain it for 15 minutes to one hour after that release the vacuum gradually using filtered (0.22-micron sterile filter) compressed air up to atmospheric pressure. The half-stoppered vials will be held at atmospheric pressure for approx. 15 minutes to one hour. 将真空度拉至 500-700 [1] mbar，达到 600 mbar 的真空度后，维持 15 分钟至 1 小时，然后使用过滤（0.22 微米无菌过滤器）压缩空气逐渐释放真空至大气压。半塞小瓶将在大气压下保持约 15 分钟至 1 小时。 [1] The minimum vacuum level observed in lyophillizer is 500 mbarA, where the no fallen rubber stopper and spillage of solution of half stoppered vials was observed.With 500 mbarA in half stoppered vials will not inhibit the microbial growth was observed. The approach of simulated load / unload with shortened hold time has been followed as per PDA Technical Report No. 22. [1] 冻干机中观察到的最低真空度为 500 mbarA，此时未观察到橡胶塞掉落和半塞小瓶溶液溢出。半塞小瓶中500mbarA不会抑制微生物生长。根据PDA技术报告第 22 号，采用缩短保持时间的模拟加载/卸载方法。 Second Pull of Vacuum 第二次抽真空Pull the vacuum to 500-700 mbar after about one hour of first pull of vacuum and after achieving the vacuum of 600 mbar and maintain it for 15 minutes to one hour after this release the vacuum gradually using filtered (0.22-micron sterile filter) compressed air up to atmospheric pressure. 首次抽真空约一小时后，将真空度拉至 500-700 mbar，达到 600 mbar 的真空度后维持 15 分钟至一小时，然后使用过滤（0.22 微米无菌过滤器）压缩空气逐渐释放真空至大气压。 The half-stoppered vials will be held at atmospheric pressure for approx.15 minutes to one hour, after this period perform the stoppering of vials into the chamber atmospheric pressure. 将半塞小瓶在大气压力下保存约 15 分钟至 1 小时，此后将小瓶塞入容器，大气压力下。 Note: In case of anaerobic media fill, break the vacuum of lyophilizer using filtered nitrogen gas, instead of filtered compressed air. 注意：如果培养基充满厌氧菌，请使用过滤氮气打破冻干机的真空，而不要使用过滤压缩空气。 How many Vials shall be loaded? 应装载多少个小瓶？1. Minimum incubation quantity of 10000 vials. Accordingly, batch size shall be designed during media fill. 1. 最小培养量为 10000 瓶。因此，应在培养基灌装时设计批次的大小。 2. Minimum number of vials to be filled shall be decided based on equipment constraint, where number of vials shall be filled considering the maximum capacity of the equipment. 2. 需要灌装的最小小瓶数量应根据设备限制决定，其中应考虑设备的最大容量来灌装小瓶。 Note: The media fill duration shall be sufficient to cover actual duration of the filling of the drug product. The leftover media after completion of the filling shall be measured and discarded. 注：培养基灌装时间应足以覆盖药品实际灌装时间。灌装完成后剩余的培养基应计量并丢弃。 Visual Inspection of filled vials 已灌装小瓶的目视检查All filled and sealed units shall be collected in sequence with respect to filling order. All the units should be inspected 100% prior to incubation. Defected vial which integrity is breached, are counted and deducted fromthe acceptable units filled. Do not reject units for low or high fill volume any cosmetic defect and other rejection, which integrity is not breached. The filled vials must be inverted or otherwise physically manipulated to ensure that all surfaces including the internal surfaces of the stopper are thoroughly wetted by the medium. 所有已灌装和密封的单元应按灌装顺序依次收集。所有单元在培养前应进行100%检查。完整性被破坏的缺陷小瓶将被计数并从已灌装的合格单元中扣除。不要因灌装量低或高、外观缺陷或其他原因而拒绝单元，只要完整性没有被破坏。已灌装的小瓶必须倒置或以其他方式进行物理操作，以确保所有表面（包括瓶塞的内表面）都被培养基彻底润湿。 Handling of online rejects (Liquid and Lyo APS) generated in the aseptic processing area during media fill run 处理培养基灌装工艺中无菌处理区域产生的在线废品（液体和Lyo APS）Units removed during an APS should not be more than units removed in routine production run. The quantity and rationale for manually or automatically removed units from the filling line must be documented. APS期间移除的单元数不应超过日常生产运行中移除的单元数。必须记录从灌装线上手动或自动移除单元的数量和理由。 Good Practices to be considered for rejects 被拒绝时应考虑的良好做法ŸThe rejects of filled units generated at all the stages of processing during media fill runs and those rejects in unstoppered state (missing or displaced stoppers) shall be considered as non-integral rejects as they are not intact (lacks integrity). These non-integral rejects shall be rejected online during media fills and shall not be considered for incubation. 在培养基灌装过程中，所有加工阶段产生的灌装单元拒收品以及未封口状态的拒收品（封口缺失或移位）应被视为非完整的拒收品，因为它们不完整（缺乏完整性）。这些非完整拒收品应在培养基灌装工艺中在线拒收，不应考虑用于培养。 ŸThe half stoppered or partially stoppered filled vials are considered as unstoppered reject units. Hence rejects of these categories during media fill runs shall be considered as non-integral rejects as they are not intact (lacks integrity). These non-integral rejects shall be rejected online during media fills and shall not be considered for incubation. 半塞或部分塞满的灌装小瓶被视为未塞住的拒收单元。因此，在培养基灌装过程中，这些类别的拒收应被视为非完整的拒收，因为它们不完整（缺乏完整性）。这些非完整拒收应在培养基灌装工艺中在线拒收，不应考虑用于培养。 ŸThe fully stoppered filled reject units generated at all the stages of processing during media fill runs to be recorded and can be incubated. This is for investigation purpose only. (in view of container closureconfiguration of aseptic products manufactured for commercialization, these rejects are considered asnon – integral units and hence are rejected and destroyed routinely). 在培养基灌装工艺中，所有加工阶段产生的完全封闭的灌装废品单元均应记录下来并进行培养。这仅用于调查目的。（鉴于为商业化生产的无菌产品的容器封闭配置，这些废品被视为非完整的单元，因此会定期被拒收和销毁）。 ŸTo enhance the assessment of contamination risks and obtain valuable information that may havepotential contamination from the simulated activities/interventions during aseptic products manufacturing, the media filled fully stoppered rejects shall be preserved in the aseptic area during media fill run. These reject units shall be subjected to sealing at the end of the media fill run and shall be incubated separately with identification details after subjecting to inspection. 为加强对污染风险的评估，并获取无菌产品生产工艺中模拟活动/干预可能造成污染的宝贵信息，在培养基灌装过程中，应将培养基灌装的完全封闭的不合格品保存在无菌区域。这些不合格品应在培养基灌装结束时密封，并在接受检查后单独培养并注明识别信息。 ŸThe fully stoppered vial with partial sealing (improper sealed unit without leak) generated during media fill runs shall be considered as non-integral rejects (in view of container closure configuration of aseptic products manufactured for commercialization, these rejects are considered as non – integral units and hence are rejected and destroyed routinely). These rejects shall be incubated separately with identification details after subjecting to inspection. 培养基灌装过程中产生的全封闭、部分密封的小瓶（密封不当且无泄漏的单元）应视为非完整的不合格品（鉴于用于商业化的无菌产品的容器封闭结构，这些不合格品被视为非完整的单元，因此日常被拒收和销毁）。这些不合格品应在接受检查后单独培养，并注明识别信息。 ŸAssess the contamination risks and obtain valuable information, the above stated rejects shall be incubated separately for information purpose only. Any positive results of these rejects shall not beconsidered for interpretation of media fill results as these units will not provide meaningful measure ofthe aseptic process and do not represent either acceptable production practices, or acceptable container closure integrity. However, failures if any (positives) in the above rejects shall be investigated separately through deviation to determine the origin of contamination and the scope of the problem if any. Ÿ评估污染风险并获取有价值的信息，上述拒收品应单独培养，仅供参考。这些拒收品的任何阳性结果均不应被视为培养基灌装结果的解释，因为这些单位不会提供无菌工艺的有意义的衡量标准，也不代表可接受的生产实践或可接受的容器封闭完整性。但是，如果上述拒收品中有任何不合格品（阳性），应通过偏差单独调查，以确定污染的来源和问题的范围（如果有）。 Part IIFollowing our previous article part I, the purpose of this white paper is to introduce Annex 1 and to describe the significant changes and the scientific/regulatory reasons behind the design of aseptic process simulation for lyophilized drug products with special considerations in key validation aspects of lyophillizer. 继我们之前的文章第一部分之后，本白皮书的目的是介绍附录1，并描述冻干药品无菌工艺模拟设计背后的重大变化和科学/监管原因，并特别考虑冻干机的关键验证方面。 Validation and Revalidation strategies for lyophilizer 冻干机的验证和再验证策略From my experience, I Would recommend dividing the qualification plans with certain frequency. Minimum is performance qualification and performance re qualification. 根据我的经验，我建议以一定的频率划分确认计划。最低限度是性能确认和性能再确认。 Routine Qualification 日常确认Frequency of routine qualification: Once in a six month 日常确认频率：每六个月一次 Some of the tests can still be relaxed to once/twice a year based on risk assessment. 根据风险评估，某些测试仍可放宽至每年一次/两次。 Performance re-qualification criteria 性能再确认标准ŸChanges in design or major maintenance 设计变更或重大维护 Ÿ Relocation of the equipment 设备搬迁 Note: For the performance re-qualification of system after changes in design or major maintenance required test should be performed based on impact of change on equipment performance. 注：对于设计变更或重大维护后系统的性能再确认，应根据变更对设备性能的影响进行所需的测试。 From my experience, Below tests shall be considered for initial qualification 根据我的经验，以下测试应考虑用于首次确认
Routine Qualification: Six Months once 日常确认：六个月一次
Routine Qualification: Two years once 日常确认：两年一次
Hold time of SIP - In lyophillizer 在冻干机中的SIP保持时间The holding time between the sterilization cycle and use should be appropriately challenged during aseptic process simulation (APS). 在无菌工艺模拟（APS）期间，应适当挑战灭菌周期和使用之间的保持时间。 Holding time after sterilization cycle till lyo door opening to load the media filled vials 灭菌循环后至冻干门打开以装入培养基灌装小瓶的保持时间 ŸNeeds to be challenged during aseptic process simulation (APS). 需要在无菌工艺模拟（APS）工艺中接受挑战。 ŸHow long? Based in process need and risk assessment. 多长时间？根据工艺需要和风险评估。 Ÿ24-36 Hours? (some companies simulated it as minimum hold duration) 24-36 小时？（一些公司模拟它作为最短保持时间） ŸLess than or equal to 36 hours is considered in GMP runs. 在GMP运行中，小于或等于36小时被认为是有效的。 Rationale for Leak Test In lyophillizer 冻干机泄漏测试的基本原理Assurance of the vacuum integrity of freeze - dryers used for the manufacture of sterile pharmaceutical products is essential for GMP operations. 确保用于生产无菌药品的冻干机的真空完整性对于 GMP 操作至关重要。 However, there is currently no generally accepted scientific rationale for establishing acceptance criteria for such testing. 然而，目前尚无普遍接受的科学原理来制定此类测试的可接受标准。 As it stands, current acceptance criteria are generally based on equipment capability – perhaps proposed by the manufacturer, or from data collected during qualification of a new freeze - dryer. The targeted specification is often one cited by The Parenteral Society. 目前，目前的可接受标准通常基于设备能力——可能是由生产商提出的，也可能是根据新冻干机确认过程中收集的数据。目标规范通常是美国肠外营养学会引用的规范。 “A frequently specified leak rate for new, clean, dry, and empty freezer dryers would be 2 x 10-2 mbar - liter/sec or 1 x 10-2 mbar - liter/sec (15 μm (mTorr) liters per sec). The leak rate should not change significantly during the life of the freeze dryer ...”. “新的、干净的、干燥的空冻干机的常见规定泄漏率为2x10-2mbar-升/秒或1x10-2mbar-升/秒（15μm(mTorr)升/秒）。在冻干机的使用寿命内，泄漏率不应发生显著变化...”。
Comparison between EU Annex-1 2008 and Annex-1 2022 欧盟附录1 2008与附录1 2022 的比较
What Annex-1- 2008 says about Lyophilization 附录1- 2008 关于冻干的规定Aseptic preparation: 34 Prior to the completion of stoppering, transfer of partially closed containers, as used in freeze drying should be done either in a grade A environment with grade B background or in sealed transfer trays in a grade B environment. 无菌准备：34 在完成加塞之前，用于冻干的部分封闭容器的转移应在具有 B 级背景的 A 级环境中进行，或在 B 级环境中的密封转移托盘中进行。 Finishing of sterile products: 116 Partially stoppered freezedrying vials should be maintained under Grade A conditions at all times until the stopper is fully inserted. 无菌产品的后处理：116 部分封闭的冻干瓶应始终保持在A级条件下，直到瓶塞完全插入。 Finishing of sterile products: 123 Containers sealed under vacuum should be tested for maintenance of that vacuum after an appropriate, pre-determined period. 无菌产品的后处理：123 真空密封的容器应在适当的预定时间后进行真空维持测试。 Annex-1 2008 附录1 2008
Annex-1 2022 附录-1 2022
Key Points from Annex-1-2022 about Lyophilization 附录1-2022中有关冻干的要点8.122 : The sterilization of the lyophilizer and associated equipment (e.g. trays, vial support rings) should be validated and the holding time between the sterilisation cycle and use appropriately challenged during APS. The lyophilizer should be sterilised regularly, based on system design. Re-sterilisation should be performed following maintenance or cleaning. Sterilised lyophilizers and associated equipment should be protected from contamination after sterilisation. 8.122：应验证冻干机和相关设备（例如托盘、小瓶支撑环）的灭菌情况，并在 APS 期间适当挑战灭菌周期与使用之间的保持时间。应根据系统设计定期对冻干机进行灭菌。维护或清洁后应再灭菌。灭菌后的冻干机和相关设备应避免受到污染。 8.123: Lyophilizers and associated product transfer and loading/unloading areas should be designed to minimize operator intervention as far as possible. The frequency of lyophilizer sterilisation should be determined based on the design and risks related to system contamination during use. Lyophilizers that are manually loaded or unloaded with no barrier technology separation should be sterilised before each load. For lyophilizers loaded and unloaded by automated systems or protected by closed barrier systems, the frequency of sterilisation should be justified and documented as part of the CCS. 8.123：冻干机及相关产品转移和装载/卸载区域的设计应尽可能减少操作员干预。冻干机灭菌频率应根据设计和使用过程中与系统污染相关的风险来确定。手动装载或卸载且无屏障技术隔离的冻干机应在每次装载前进行灭菌。对于由自动化系统装载和卸载或受封闭屏障系统保护的冻干机，应证明灭菌频率的合理性并将其记录为 CCS 的一部分。 Explaining 8.123 : Sterilization in Lyophillizer and its Hold time simulations 解释 8.123：冻干机灭菌及其保持时间模拟 A generally recognized acceptable method of sterilizing the lyophilizer is through the use of moist steam under pressure. For the sterilisation process, saturated steam is introduced into the freeze-drying system. 普遍认可的冻干机灭菌方法是使用加压的湿蒸汽。在灭菌过程中，将饱和蒸汽引入冻干系统。 In the sterilisation phase, the chamber, and the condenser along with all connected piping (including venting filters) are sterilised. The pressure is controlled by means of the steam pressure regulators according to the chamber pressure measurement. Due to high pressure requirements during steam sterilization, manufacturers must design, test, and approve both chamber, condenser as rated pressure vessel. Additionally, there are numerous requirements for other factors such as pipes lengths, bends, filters, that engineers must consider as steam must penetrate all areas of the freeze dryer in order to serve as an effective sterilizing agent. 在灭菌阶段，灭菌室、冷凝器以及所有连接的管道（包括通风过滤器）均需灭菌。根据灭菌室压力测量值，通过蒸汽压力调节器控制压力。由于蒸汽灭菌期间对压力要求较高，生产商必须设计、测试和批准灭菌室和冷凝器作为额定压力容器。此外，工程师还必须考虑许多其他因素，例如管道长度、弯头、过滤器等，因为蒸汽必须渗透到冻干机的所有区域才能作为有效的灭菌剂。 The sterilisation temperature is measured at the following drainage points: Chamber outlet, Condenser Outlet, CIP Pump Outlet and Filter outlet. 在以下排水点测量灭菌温度：灭菌室出口、冷凝器出口、CIP泵出口和过滤器出口。 At the end of the sterilisation process, the freeze-drying system is dried with the aid of the liquid-ring pump. The drying shelf assembly and the chamber wall are re-cooled after drying. 灭菌过程结束时，冻干系统在液环泵的帮助下进行干燥。干燥架组件和腔壁在干燥后再冷却。 The sterilisation of the lyophilizer and filling line associated equipment should be validated, and sterilization frequency should be justified. 应验证冻干机和灌装线相关设备的灭菌情况，并证明灭菌频率合理。 The lyophilizer chamber is to be Cleaned and sterilized between batches because of the direct means of contamination. (Expectation of cleaning and sterilization is also applicable when there is maintenance in lyophillizer). 由于直接污染途径，批次之间需要对冻干机腔体进行清洁和灭菌。（当冻干机进行维护时，清洁和灭菌的期望也适用）。 The holding time between the sterilisation cycle and its use should be appropriately challenged during aseptic process simulation (APS). 在无菌工艺模拟（APS）期间，应适当挑战灭菌周期与使用之间的保持时间。 8.124 The integrity of the lyophilizer should be maintained following sterilisation and during lyophilization. The filter used to maintain lyophilizer integrity should be sterilised before each use of the system and its integrity testing results should be part of the batch certification/release. The frequency of vacuum/leak integrity testing of the chamber should be documented and the maximum permitted leakage of air into the lyophilizer should be specified and checked at the start of every cycle. 8.124 应在灭菌后和冻干过程中保持冻干机的完整性。用于保持冻干机完整性的过滤器应在每次使用系统前进行灭菌，其完整性测试结果应作为批次确认/放行的一部分。应记录腔体真空/泄漏完整性测试的频率，并应规定和检查每个循环开始时允许的最大空气泄漏量。 8.125 Lyophilization trays should be checked regularly to ensure that they are not misshapen or damaged. 8.125 应定期检查冻干托盘，确保其没有变形或损坏。 8.126 Points to consider for the design of loading (and unloading, where the lyophilised material is still unsealed and exposed), include but are not limited to: 8.126 装载设计（以及卸载，其中冻干材料仍未密封并暴露）时需要考虑的要点包括但不限于： i. The loading pattern within the lyophilizer should be specified and documented. i. 应指定并记录冻干机内的装载模式。 ii. The transfer of partially closed containers to a lyophilizer should be undertaken under grade A conditions at all times and handled in a manner designed to minimize direct operator intervention. Technologies such as conveyor systems or portable transfer systems (e.g. clean air transfer carts, portable unidirectional airflow workstations) should be used to ensure that the cleanliness of the system used to transfer the partially closed containers is maintained. Alternatively, where supported by validation, trays closed in grade A and not reopened whilst in the grade B area may be used to protect partially stoppered vials (e.g. appropriately closed boxes). ii. 将部分封闭的容器转移到冻干机时，应始终在 A 级条件下进行，并以尽量减少操作员直接干预的方式进行处理。应使用传送系统或便携式转移系统（例如洁净空气转移车、便携式单向气流工作站）等技术来确保用于转移部分封闭容器的系统的清洁度得到维持。或者，如果得到验证的支持，可以使用在 A 级条件下关闭且在 B 级区域内未再打开的托盘来保护部分封闭的小瓶（例如适当封闭的盒子）。 iii. Airflow patterns should not be adversely affected by transport devices and venting of the loading zone. iii. 气流模式不应受到传输设备和装载区通风的不利影响。 iv. Unsealed containers (such as partially stoppered vials) should be maintained under grade A conditions and should normally be separated from operators by physical barrier technology or any other appropriate measures. iv. 未密封的容器（如部分封闭的小瓶）应保持在 A 级条件下，并且通常应通过物理屏障技术或任何其他适当措施与操作员隔开。 v. Where seating of the stoppers is not completed prior to opening the lyophilizer chamber, product removed from the lyophilizer should remain under grade A conditions during subsequent handling. v. 如果在打开冻干机腔体之前未完成压塞，则从冻干机中取出的产品在后续处理工艺中应保持在A级条件下。 vi. Utensils used during loading and unloading of the lyophilizer (e.g. trays, bags, placing devices, tweezers) should be sterile. vi. 冻干机装卸工艺中使用的器具（如托盘、袋子、放置装置、镊子）应是无菌的。 Conclusion 结论Lyophilization is a critical process step and all activities that can affect the sterility of the product or material need to be regarded as extensions of the aseptic processing of the sterilised product. The lyophilization equipment and its processes should be designed to ensure that product or material sterility is maintained during lyophilization by preventing microbial and particle contamination between the filling of products for lyophilization, and completion of lyophilization process. All control measures in place should be determined by the site’s CCS. 冻干是关键的工艺步骤，所有可能影响产品或材料无菌性的活动都应视为灭菌产品无菌处理的延伸。冻干设备及其工艺应设计为确保在冻干工艺中保持产品或材料的无菌性，防止在产品灌装冻干和冻干工艺完成之间发生微生物和颗粒污染。所有控制措施均应由场地的 CCS 确定。 From Lyo APS Design point of view,the duration of the media filling stated above represents overnight lyophilization cycle. We ensure the integrity of the lyophilizer chamber by testing the lyophilizer post SIP cycle, therefore no additional benefit is drawn by holding the filled vials for longer duration since the lyophilizer’s integrity is maintained throughout cycle. 从冻干APS设计的角度来看，上述培养基灌装时间代表隔夜冻干周期。我们通过在SIP循环后测试冻干机来确保冻干机室的完整性，因此，将已灌装的小瓶保存更长的时间不会带来额外的好处，因为冻干机的完整性在整个周期中都得到保持。 Hence, it is not necessary to carry out lyophilization cycle as per the actual drug product lyophilization cycle. As compared to lyophilization hold period, probability of contamination of vials is more during the process stages such as filling, stoppering etc and also As per regulations from few regulatory authorities it’s clear that hold time does not need to be the actual duration of lyophilization cycle. 因此，没有必要按照实际药品冻干周期进行冻干周期。与冻干保持期相比，在灌装、封盖等工艺阶段，小瓶被污染的可能性更大，而且根据一些监管机构的规定，保持时间不必是实际的冻干周期持续时间。 There should be written justification for the hold duration of media fill vials in the aseptic simulation process of freeze drying process. 在冻干过程的无菌模拟工艺中，应对培养基灌装瓶的保持时间提供书面说明。 Hence, A balance risk and science-based approach is needed to simulate the process as closely as possible, and rationale for holding the vials in the lyophillizer need to be presented which is the expectation from the regulators. 因此，需要一种平衡风险和基于科学的方法来尽可能紧密地模拟该过程，并且需要提供将小瓶保持在冻干机中的理由，这是监管机构的期望。 Finally in case of failure in the media it necessary to diagnose and prove the source of contamination accurately so that robust corrective or preventive actions get implemented. 最后，如果培养基出现失败，需要准确诊断并证明污染源，以便实施强有力的纠正或预防措施。 |
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