培養(yǎng)基模擬灌裝試驗講解
發(fā)布時間：
2022-12-27
作者：
無菌檢驗培養(yǎng)基
評價一個無菌工藝的操作能力最有用的方法是工藝模擬試驗，及培養(yǎng)基模擬灌裝試驗。過往對培養(yǎng)基模擬灌裝試驗僅僅關(guān)注無菌灌裝階段，沒有對可能影響無菌終產(chǎn)品質(zhì)量的無菌操作工藝評價，因此現(xiàn)行培養(yǎng)基模擬灌裝試驗主要目的是評估在既定無菌生產(chǎn)環(huán)境和過程控制條件下生產(chǎn)無菌產(chǎn)品的能力、證明指定的無菌工藝設(shè)計和變更是否可行、證明無菌工藝過程中的相關(guān)操作是否可行、評估無菌工藝人員的操作水平、發(fā)現(xiàn)無菌工藝過程中潛在的微生物污染因素、以及對現(xiàn)行GMP要求的合規(guī)能力。
培養(yǎng)基模擬灌裝應(yīng)盡可能地模擬產(chǎn)品本身所經(jīng)歷的與設(shè)備接觸的表面、容器密封系統(tǒng)、關(guān)鍵環(huán)節(jié)和關(guān)鍵工藝操作的接觸，根據(jù)產(chǎn)品的生產(chǎn)工藝類型，確認(rèn)無菌生產(chǎn)工藝的模擬灌裝操作步驟，因凍干粉針劑無菌生產(chǎn)工藝是相對齊全的無菌生產(chǎn)工藝，下面以此為例，簡介一下培養(yǎng)基模擬灌裝試驗的關(guān)注點。
培養(yǎng)基模擬灌裝試驗主要應(yīng)關(guān)注以下內(nèi)容但不是全部，生產(chǎn)線的狀態(tài)、試驗的頻率、培養(yǎng)基類型、灌裝持續(xù)時間、灌裝的數(shù)量、容器（西林瓶）體積、生產(chǎn)線速度、灌裝體積、挑戰(zhàn)性試驗、培養(yǎng)、結(jié)果與評級、清場和清洗、模擬灌裝中的有效期等。
生產(chǎn)線的狀態(tài)和試驗的頻率，新生產(chǎn)線確認(rèn)期間每個班次至少執(zhí)行3次連續(xù)的符合要求的試驗；持續(xù)性生產(chǎn)線按照每班次和工藝每年重復(fù)2次；生產(chǎn)線在退役前，進行一次無菌灌裝；以下變更情況需要進行再驗證，生產(chǎn)線發(fā)生重大改變、直接接觸產(chǎn)品的設(shè)備、容器改變、參與生產(chǎn)的主要生產(chǎn)人員改變。
培養(yǎng)基類型應(yīng)基于產(chǎn)品的劑型，選擇的培養(yǎng)基應(yīng)能夠支持較寬泛的微生物，最好能符合內(nèi)部種屬；澄清度應(yīng)以便于輕松的觀察到渾濁為宜、濃度應(yīng)當(dāng)遵循供應(yīng)商建議的濃度、若無菌生產(chǎn)過程中使用了過濾器，培養(yǎng)基應(yīng)能夠通過生產(chǎn)過程同一級別的過濾器過濾為準(zhǔn)，如需稀釋過濾，培養(yǎng)基濃度應(yīng)提供微生物生長能力驗證；同時培養(yǎng)基料液應(yīng)能滿足全部覆蓋生產(chǎn)設(shè)備。
灌裝持續(xù)時間對于初始性驗證，培養(yǎng)基灌封實驗應(yīng)安排在一周內(nèi)的不同時間進行；對于再驗證，可選擇在一周結(jié)束或其它最差條件下進行；灌封時間要涵蓋實際生產(chǎn)班次；持續(xù)時間應(yīng)根據(jù)設(shè)置操作和干預(yù)活動所需時間，以及實際無菌操作時間來確定；大批量模擬灌裝時，可使用一些空白樣品（空的或灌水）以維持模擬實驗的動態(tài)環(huán)境；人工灌裝或密封操作的持續(xù)時間應(yīng)不短于實際生產(chǎn)工藝整個流程；持續(xù)試驗應(yīng)涵蓋凍干工藝中，模擬未密封的容器置于部分抽真空的腔體，注意避免導(dǎo)致培養(yǎng)基沸騰、冷凍或凍干步驟，確保培養(yǎng)基保持在好氧的狀態(tài)以降低微生物的抑制性。
灌裝的數(shù)量的影響因素有實際批量、干預(yù)操作的類型、數(shù)量、時間、開放/隔離廠房的類型，應(yīng)當(dāng)足以模擬商業(yè)化生產(chǎn)條件并評估商業(yè)化批次污染的可能性，小批次應(yīng)當(dāng)至少等于產(chǎn)品數(shù)量，指南未對最大批次（≥5000支）做出規(guī)定，通?？山邮艿钠瘘c是5000至10000支。
容器（西林瓶）體積應(yīng)當(dāng)考慮灌裝容器的極端尺寸（最大容器和最小容器），最初的試驗中，可以選用二次最大容器，第三次選用最小容器。最大容器（通常由于灌裝量較大而導(dǎo)致最慢的灌裝速度），通常開口較大，所以環(huán)境中微生物侵入的潛在風(fēng)險較大；最小容器（通常由于灌裝量較小而導(dǎo)致最快的灌裝速度），體現(xiàn)了最大的操作難度；小的容器容易破碎，穩(wěn)定性較差，在設(shè)備中更容易破裂和堵塞；日常的評估中，其他尺寸的容器也應(yīng)包括在驗證計劃中；不能使用琥珀色的瓶子，而應(yīng)使用透明瓶子，以便可以目視檢查到微生物的生長。
生產(chǎn)線速度應(yīng)包括實際生產(chǎn)過程的灌裝速度范圍，灌裝生產(chǎn)線的首次驗證中，一次可以選用最低速度，另外兩次可以選用最高速度；正常的培養(yǎng)基模擬灌裝應(yīng)包含了最大和最小的生產(chǎn)速度。
灌裝體積不需要與現(xiàn)實中常規(guī)裝量一致；但應(yīng)有足夠的灌裝量，可以充分接觸到容器的密封件的表面，并且足以目視檢測到微生物的培養(yǎng)后的生長情況。
挑戰(zhàn)性試驗基于日常生產(chǎn)的干擾，如工作班次輪換、監(jiān)測活動、灌裝線裝配、稱量調(diào)節(jié)、加膠塞、處理倒瓶、取樣、環(huán)境監(jiān)測等日常干擾；設(shè)備故障、灌裝線堵塞、膠塞堵塞、軌道調(diào)節(jié)、拆卸/替換破損的部件等非日常干擾。
培養(yǎng)時容器必須倒置或旋轉(zhuǎn)確保所有表面，包括容器內(nèi)表面，完全接觸培養(yǎng)基；對灌有培養(yǎng)基的容器進行培養(yǎng)后目視檢查其微生物的生長；應(yīng)經(jīng)過批準(zhǔn)的規(guī)程檢查受污染安瓿的完整性以了解是否有容器或密封件損壞的證據(jù)；目檢應(yīng)該由有資質(zhì)的人員進行
結(jié)果范圍應(yīng)符合相關(guān)法規(guī)要求；結(jié)果評價對可確定污染來源時，不管是否超出了警戒線或行動限，都應(yīng)該將所有收到的污染的微生物至少標(biāo)識至屬，最好標(biāo)識至種；對于所有試驗規(guī)模，微生物污染的間歇性事故可能暗示存在有應(yīng)當(dāng)進行調(diào)查的低級別污染。對嚴(yán)重的失效的調(diào)查應(yīng)該包括對自從上一次成功的培養(yǎng)基灌裝以來生產(chǎn)的批次的無菌保證影響。
清場和清洗應(yīng)當(dāng)在培養(yǎng)基灌裝結(jié)束后采取妥善措施保證培養(yǎng)基灌裝驗證不會對后續(xù)的生產(chǎn)帶入污染的風(fēng)險；培養(yǎng)基有合適的處理方法。
培養(yǎng)基灌裝實施中效期應(yīng)關(guān)注灌裝或分裝設(shè)備、部件、儲罐、無菌物料、藥液或藥粉在實際灌裝前能夠放置的最長時間，采用最長允許儲存時間的灌裝設(shè)備、灌裝部件、儲罐、無菌物料參與培養(yǎng)基灌裝；采用無菌過濾后存放在儲罐內(nèi)的培養(yǎng)基到實際生產(chǎn)時產(chǎn)品的最大儲存時間后再灌裝。
培養(yǎng)基模擬灌裝驗證僅僅是無菌工藝系統(tǒng)在某一個時間點的表現(xiàn)，包括環(huán)境、設(shè)備、程序和人員，并不能保證不同時間段內(nèi)也可以表現(xiàn)出同樣的微生物保證水平，因此為保障無菌產(chǎn)品生產(chǎn)，通過控制和驗證所有的相關(guān)工藝，例如環(huán)境監(jiān)測、人員確認(rèn)和清潔滅菌驗證等，才有可能維持無菌工藝的水平，所以單獨驗證相關(guān)的的清潔和滅菌工藝對保障無菌工藝生產(chǎn)的中產(chǎn)品質(zhì)量也非常重要。
作者：殷志勇 來源：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.