培养基模拟灌装试验讲解

发布时间:

2022-12-27

作者:

无菌检验培养基


评价一个无菌工艺的操作能力最有用的方法是工艺模拟试验,及培养基模拟灌装试验。过往对培养基模拟灌装试验仅仅关注无菌灌装阶段,没有对可能影响无菌终产品质量的无菌操作工艺评价,因此现行培养基模拟灌装试验主要目的是评估在既定无菌生产环境和过程控制条件下生产无菌产品的能力、证明指定的无菌工艺设计和变更是否可行、证明无菌工艺过程中的相关操作是否可行、评估无菌工艺人员的操作水平、发现无菌工艺过程中潜在的微生物污染因素、以及对现行GMP要求的合规能力。
 
培养基模拟灌装应尽可能地模拟产品本身所经历的与设备接触的表面、容器密封系统、关键环节和关键工艺操作的接触,根据产品的生产工艺类型,确认无菌生产工艺的模拟灌装操作步骤,因冻干粉针剂无菌生产工艺是相对齐全的无菌生产工艺,下面以此为例,简介一下培养基模拟灌装试验的关注点。
 
培养基模拟灌装试验主要应关注以下内容但不是全部,生产线的状态、试验的频率、培养基类型、灌装持续时间、灌装的数量、容器(西林瓶)体积、生产线速度、灌装体积、挑战性试验、培养、结果与评级、清场和清洗、模拟灌装中的有效期等。
 
生产线的状态和试验的频率,新生产线确认期间每个班次至少执行3次连续的符合要求的试验;持续性生产线按照每班次和工艺每年重复2次;生产线在退役前,进行一次无菌灌装;以下变更情况需要进行再验证,生产线发生重大改变、直接接触产品的设备、容器改变、参与生产的主要生产人员改变。
 
培养基类型应基于产品的剂型,选择的培养基应能够支持较宽泛的微生物,最好能符合内部种属;澄清度应以便于轻松的观察到浑浊为宜、浓度应当遵循供应商建议的浓度、若无菌生产过程中使用了过滤器,培养基应能够通过生产过程同一级别的过滤器过滤为准,如需稀释过滤,培养基浓度应提供微生物生长能力验证;同时培养基料液应能满足全部覆盖生产设备。
 
灌装持续时间对于初始性验证,培养基灌封实验应安排在一周内的不同时间进行;对于再验证,可选择在一周结束或其它最差条件下进行;灌封时间要涵盖实际生产班次;持续时间应根据设置操作和干预活动所需时间,以及实际无菌操作时间来确定;大批量模拟灌装时,可使用一些空白样品(空的或灌水)以维持模拟实验的动态环境;人工灌装或密封操作的持续时间应不短于实际生产工艺整个流程;持续试验应涵盖冻干工艺中,模拟未密封的容器置于部分抽真空的腔体,注意避免导致培养基沸腾、冷冻或冻干步骤,确保培养基保持在好氧的状态以降低微生物的抑制性。
 
灌装的数量的影响因素有实际批量、干预操作的类型、数量、时间、开放/隔离厂房的类型,应当足以模拟商业化生产条件并评估商业化批次污染的可能性,小批次应当至少等于产品数量,指南未对最大批次(≥5000支)做出规定,通常可接受的起点是5000至10000支。
 
容器(西林瓶)体积应当考虑灌装容器的极端尺寸(最大容器和最小容器),最初的试验中,可以选用二次最大容器,第三次选用最小容器。最大容器(通常由于灌装量较大而导致最慢的灌装速度),通常开口较大,所以环境中微生物侵入的潜在风险较大;最小容器(通常由于灌装量较小而导致最快的灌装速度),体现了最大的操作难度;小的容器容易破碎,稳定性较差,在设备中更容易破裂和堵塞;日常的评估中,其他尺寸的容器也应包括在验证计划中;不能使用琥珀色的瓶子,而应使用透明瓶子,以便可以目视检查到微生物的生长。
 
生产线速度应包括实际生产过程的灌装速度范围,灌装生产线的首次验证中,一次可以选用最低速度,另外两次可以选用最高速度;正常的培养基模拟灌装应包含了最大和最小的生产速度。
 
灌装体积不需要与现实中常规装量一致;但应有足够的灌装量,可以充分接触到容器的密封件的表面,并且足以目视检测到微生物的培养后的生长情况。
 
挑战性试验基于日常生产的干扰,如工作班次轮换、监测活动、灌装线装配、称量调节、加胶塞、处理倒瓶、取样、环境监测等日常干扰;设备故障、灌装线堵塞、胶塞堵塞、轨道调节、拆卸/替换破损的部件等非日常干扰。
 
培养时容器必须倒置或旋转确保所有表面,包括容器内表面,完全接触培养基;对灌有培养基的容器进行培养后目视检查其微生物的生长;应经过批准的规程检查受污染安瓿的完整性以了解是否有容器或密封件损坏的证据;目检应该由有资质的人员进行
结果范围应符合相关法规要求;结果评价对可确定污染来源时,不管是否超出了警戒线或行动限,都应该将所有收到的污染的微生物至少标识至属,最好标识至种;对于所有试验规模,微生物污染的间歇性事故可能暗示存在有应当进行调查的低级别污染。对严重的失效的调查应该包括对自从上一次成功的培养基灌装以来生产的批次的无菌保证影响。
 
清场和清洗应当在培养基灌装结束后采取妥善措施保证培养基灌装验证不会对后续的生产带入污染的风险;培养基有合适的处理方法。
 
培养基灌装实施中效期应关注灌装或分装设备、部件、储罐、无菌物料、药液或药粉在实际灌装前能够放置的最长时间,采用最长允许储存时间的灌装设备、灌装部件、储罐、无菌物料参与培养基灌装;采用无菌过滤后存放在储罐内的培养基到实际生产时产品的最大储存时间后再灌装。
 
培养基模拟灌装验证仅仅是无菌工艺系统在某一个时间点的表现,包括环境、设备、程序和人员,并不能保证不同时间段内也可以表现出同样的微生物保证水平,因此为保障无菌产品生产,通过控制和验证所有的相关工艺,例如环境监测、人员确认和清洁灭菌验证等,才有可能维持无菌工艺的水平,所以单独验证相关的的清洁和灭菌工艺对保障无菌工艺生产的中产品质量也非常重要。
 
作者:殷志勇 来源:蒲公英
 

 
The most useful methods to evaluate the operational capability of a aseptic process are process simulation test and culture medium simulation filling test. Past to medium filling simulation test focus on aseptic filling stage, not on the quality of end products may affect the sterile sterile operation evaluation process, so the current medium filling simulation test main purpose is to assess in established under the condition of aseptic production environment and process control in the production of sterile product ability, to prove that specify the aseptic process design and change is feasible, prove whether related operations in the process of aseptic technique, evaluate sterile technique personnel operating level, found sterile potential microbial contamination factors in the process, as well as to the current GMP compliance.
 
Medium filling simulation should be as much as possible to simulate the surface of the experience of contact with the equipment by the product itself, container sealing system, key links and key operation contact, depending on the type of product the production process of confirmation of aseptic production process simulation filling operation steps, for producing injection asepsis production process is relatively complete aseptic production process, the following order, for example, an overview of the medium filling simulation test of concerns.
 
Medium filling simulation test mainly focus on the following content but not all, of the state of the production line, test duration, frequency, the types of culture medium, filling volume, the number of containers (schering bottles), line speed, filling volume, challenging test, training, results and rating, clear and clean, the validity of simulation of filling and so on.
 
The status of the production line and the frequency of the test, and at least three consecutive tests in accordance with the requirements of each shift during the confirmation of the new production line; The continuous production line is repeated twice a year according to each shift and process. The production line shall carry out a sterile filling before decommissioning. The following changes need to be re-verified. Major changes have taken place in the production line, direct contact with the product equipment, container changes, and changes in the main production personnel involved in the production.
 
The type of culture medium should be based on the dosage form of the product. The selected culture medium should be able to support a wide range of microbes, preferably in line with the internal species. Clarity should be observed in order to easily turbid advisable, concentration shall follow the supplier recommended concentration, sterile filter was used in the production process, if the medium should be able to through the production process is the same level of the filter, if you need dilution filter, medium concentration microbial growth ability verification should be provided; At the same time, the culture of base material liquid should be able to meet the full coverage of production equipment.
 
For the initial verification of the filling duration, the filling experiment of culture medium should be arranged at different times in a week. For re-verification, it can be carried out at the end of a week or under other worst conditions. The filling time should cover the actual production shift; The duration shall be determined according to the time required for setting up the operation and intervention activities, as well as the actual sterile operation time. When simulating filling in large quantities, some blank samples (empty or filled with water) can be used to maintain the dynamic environment of simulation experiments. The duration of manual filling or sealing operations shall not be shorter than the entire process of the actual production process. Continuous test should cover the lyophilization process, the simulation is not sealed container at part of the vacuum chamber, avoid boiling, frozen or dried steps lead to medium, to ensure that the medium in the aerobic condition in order to reduce the microbial inhibitory.
 
Filling the influence factors of the number of actual batch, intervention operation type, quantity, time, open/isolation plant type, should be enough to simulate commercial production conditions and evaluate the possibility of contamination commercial batches, small batches should be at least equal to the product quantity, guide to stipulates the biggest batch (5000 or higher), usually acceptable starting point is 5000 to 10000.
 
Container volume (schering bottles) should consider the extreme of filling container size (the largest container and minimum container), the initial experiment, can choose the second largest container, the third time to choose minimum container. The largest container (usually the slowest filling speed due to the large filling volume) usually has a large opening, so the potential risk of microbial invasion in the environment is greater. The smallest container (usually the fastest filling speed due to the small amount of filling) represents the most difficult operation. Small container is easy to break, less stable, more easy to break and plug in the equipment; Other sizes of containers should also be included in the validation plan in the daily evaluation; Amber bottles should not be used, but transparent bottles should be used so that the growth of microorganisms can be visually detected.
 
The production line speed shall include the range of filling speed in the actual production process. In the first verification of the filling line, the lowest speed can be selected at one time and the highest speed can be selected at the other two times. Normal medium simulated filling should include maximum and minimum production speed.
 
The filling volume does not need to be consistent with the actual conventional loading volume. However, there should be enough filling capacity to reach the surface of the sealing parts of the container and to visually detect the growth of microorganisms after culture.
 
Challenging test based on the interference of daily production, such as work shifts, monitoring activities, filling line assembly, weighing regulation, bottle stopper, handle down, sampling, environmental monitoring and other daily interference; Equipment failure, filling line blockage, rubber plug blockage, track adjustment, removal/replacement of damaged parts and other non-routine interference.
 
During culture, the container must be inverted or rotated to ensure that all surfaces, including the inner surface of the container, are fully exposed to the medium. The growth of microorganism was examined visually after culture of the container with culture medium. The integrity of the contaminated ampoule should be checked by approved procedures to see if there is evidence of damage to the container or seal; Inspection should be conducted by qualified personnel
The results should meet the requirements of relevant laws and regulations. Results when determining the source of contamination, no matter whether it is beyond the warning line or the action limit, all contaminated microorganisms received should be identified at least to the genus and preferably to the species. For all test sizes, intermittent incidents of microbial contamination may indicate the existence of low-level contamination that should be investigated. The investigation of serious failures should include the effect of aseptic guarantees on batches produced since the last successful culture medium filling.
 
Cleaning and cleaning should take proper measures after the completion of the filling of the medium to ensure that the filling verification of the medium will not bring the risk of contamination to the subsequent production. The medium has an appropriate treatment.
 
Effect in the implementation of media fills should focus on filling and packing equipment, components, storage tank, sterile material, liquid or powder before the actual filling can be placed for the longest time, and the longest allows the storage time of filling equipment, filling components, storage tank, aseptic filling material in culture medium; The culture medium stored in the storage tank after sterile filtration is adopted, and the maximum storage time of the product at the actual production is then filled.
 
Medium filling simulation verification is only sterile process system at a certain point in time, including environment, equipment, procedures and personnel, there is no guarantee that different time period can also show the same level of microbial guarantee, so to protect aseptic production, through the control and validation of all the related process, such as environment monitoring, personnel qualification and clean the sterilization validation, etc., is likely to maintain levels of aseptic process, so a separate validation related cleaning and sterilization process to ensure product quality in the production of sterile is also very important.