mRNA治療的臨床潛力分析與應用
信使RNA（mRNA）是一類新興的生物治療藥物。雖然它們有相當大的前景，但這一領域仍處于起步階段。mRNA治療為挑戰性疾病的靶向治療和靈活生產提供了新的機遇，抗COVID-19的mRNA疫苗的快速發展就證明了這一點。
mRNA的臨床潛力已經超出了疫苗的范疇，擴展到其他先進的治療方法，例如蛋白質替代、基因治療和癌癥免疫治療。
由于mRNA是脆弱的，不穩定的，開發 mRNA 疫苗的最重要的挑戰仍然是其固有的不穩定性，因為它很有可能在冰點以上分解。
mRNA 構建模塊的修飾以及能夠相對安全地包裹它顆粒的開發幫助了mRNA 疫苗候選者。但是這種新型疫苗仍然需要前所未有的冷凍條件存儲、運輸和管理。
WHO ：
在用 LNP 包封期間以及制造條件下應特別注意盡量減少 mRNA 的降解，這個包封和生產添加會影響 LNP 及最終 mRNA-LNP 疫苗產品的穩定性，例如：產生的影響條件包括mRNA解凍和LNPs或mRNA LNPs的冷凍速率。
Assessment report---COVID-19 Vaccine（根據某上市的mRNA疫苗的評估報告---EMA）
Characterisation 特征 ---提供了凍融降解的數據
The applicant provided a detailed characterisation of the mRNA active substance including detailed information on the structural properties of the mRNA CX-024414. Additionally, data on the chromatographic profile, the thermal denaturation as well as freeze thaw degradation and their influence on protein expression have been provided.
申請人提供了mRNA活性物質的詳細特征，包括mRNA CX-024414結構特性的詳細信息。此外，還提供了色譜圖、熱變性和凍融降解及其對蛋白質表達的影響數據。
The composition of the finished product
最終產品的成分---采用蔗糖來提高凍融和存儲穩定性
Sucrose is added to promote product stability through freeze/thaw and long-term storage.
添加蔗糖是為了通過冷凍/解凍和長期儲存來提高產品的穩定性。
Manufacturing process development
生產工藝開發---增加了放大和大體積冷凍步驟
mRNA-loaded LNP intermediate 載有 mRNA 的 LNP 中間體
The product consists of an mRNA-lipid complex dispersion that contains the mRNA (CX-024414) that encodes for the pre-fusion stabilised Spike protein of 2019-novel Coronavirus (SARS-CoV-2).Comprehensive development data have been provided. Changes made during development have been described. They included scale-up, addition of a bulk freezing step, change of some equipment and revision of the lipid molar ratios to allow for harmonisation with the platform process.
該產品由一種mRNA-脂質復合分散體組成，其中包含編碼2019年新型冠狀病毒（SARS-CoV-2）融合前穩定 Spike 蛋白的mRNA（CX-024414）。已提供全面的開發數據。已經描述了在開發過程中所做的變更。它們包括放大、增加批量冷凍步驟、改變一些設備和修改脂質摩爾比以與平臺工藝匹配。
Manufacture of the product and process controls
產品生產和工藝控制---涵蓋了冷凍工藝
The LNP manufacturing process comprises lipid stock solution (LSS) preparation, nanoprecipitation mixing, tangential flow filtration (TFF), dilution and cryoprotectant addition, clarification, fill, and freezing and storage.
LNP 制造過程包括脂質儲備溶液 (LSS) 制備、納米沉淀混合、切向流過濾 (TFF)、稀釋和冷凍保護劑添加、澄清、填充以及冷凍和儲存。
Stability of the product
產品的穩定性--現有的凍融工藝對產品的穩定性沒有影響
mRNA-loaded LNP intermediate載有 mRNA 的 LNP 中間體
Stability studies in support of mRNA-loaded LNP intermediate freeze-thaw cycling were performed using the development lot. The results show that the mRNA-1273 LNP biophysical attributes (particle size, polydispersity, encapsulation) are not impacted by the different freeze-thaw processes (room temperature or 2°C to 8°C) or 5 cycles of freeze-thaw
使用開發批次進行了支持負載 mRNA 的 LNP 中間凍融循環的穩定性研究。結果表明，mRNA-1273 LNP 生物物理屬性（粒徑、多分散性、封裝）不受不同凍融過程（室溫或 2°C 至 8°C）或 5 個凍融循環的影響，還進行了支持成品凍融循環的穩定性研究。凍融循環最多重復五次。所有結果都在規格范圍內，表明最終產品在重復凍融步驟方面相對穩定，支持五個凍融循環。然而，批準的產品信息中不允許在上市時反復凍融小瓶。
Finished product
最終成品---仍然需要做凍融測試
Stability studies in support of finished product freeze-thaw cycling were also performed. The freeze-thaw cycling was repeated up to five times. All results were within the specification and indicate that the final product is relatively stable with regard to repeated freeze-thaw steps support five freeze/thaw cycles. Repeated freeze-thaw of vials upon marketing is however, not permitted in the approved product information
Design and conduct of clinical studies
臨床研究的設計和執行---仍需要評估凍融的穩定性，涵蓋運輸和存儲
The RT-PCR assay is acceptably validated and additional validation studies to evaluate stability of clinical samples at various temperatures and freeze/thawing cycles to cover shipping and storage conditions were provided.
RT-PCR 測定得到了可接受的驗證，并提供了額外的驗證研究，以評估臨床樣品在各種溫度和冷凍/解凍循環中的穩定性，以涵蓋運輸和儲存條件。
Why not impacted by the different freeze-thaw processes
為什么凍融工藝對產品沒有影響?
凍融工藝的開發
特殊要求的凍融曲線降低了對mRNA產品的影響
The goal must be to reduce STRESSES DURING FREEZING
目標必須是在凍結過程中減少壓力
Stress is coursed by:
壓力主要這幾方面
• CRYOCONCENTRATION冷凍濃縮
• PH SHIFT pH pH漂移
• PROTEIN INTERACTION WITH THE ICE SURFACE蛋白冰液界面的變性
mRNA Freezing experience
mRNA冷凍實驗
mRNA experienced companies are freezing as fast as possible, so with rates from + to -60° C in < 5hours
經驗豐富的mRNA公司會盡可能快地凍結mRNA（在< 5小時內從+到-60°C凍結）
mRNA的臨床潛力已經超出了疫苗的范疇，擴展到其他先進的治療方法，例如蛋白質替代、基因治療和癌癥免疫治療。
由于mRNA是脆弱的，不穩定的，開發 mRNA 疫苗的最重要的挑戰仍然是其固有的不穩定性，因為它很有可能在冰點以上分解。
mRNA 構建模塊的修飾以及能夠相對安全地包裹它顆粒的開發幫助了mRNA 疫苗候選者。但是這種新型疫苗仍然需要前所未有的冷凍條件存儲、運輸和管理。
WHO ：
在用 LNP 包封期間以及制造條件下應特別注意盡量減少 mRNA 的降解，這個包封和生產添加會影響 LNP 及最終 mRNA-LNP 疫苗產品的穩定性，例如：產生的影響條件包括mRNA解凍和LNPs或mRNA LNPs的冷凍速率。
Assessment report---COVID-19 Vaccine（根據某上市的mRNA疫苗的評估報告---EMA）
Characterisation 特征 ---提供了凍融降解的數據
The applicant provided a detailed characterisation of the mRNA active substance including detailed information on the structural properties of the mRNA CX-024414. Additionally, data on the chromatographic profile, the thermal denaturation as well as freeze thaw degradation and their influence on protein expression have been provided.
申請人提供了mRNA活性物質的詳細特征，包括mRNA CX-024414結構特性的詳細信息。此外，還提供了色譜圖、熱變性和凍融降解及其對蛋白質表達的影響數據。
The composition of the finished product
最終產品的成分---采用蔗糖來提高凍融和存儲穩定性
Sucrose is added to promote product stability through freeze/thaw and long-term storage.
添加蔗糖是為了通過冷凍/解凍和長期儲存來提高產品的穩定性。
Manufacturing process development
生產工藝開發---增加了放大和大體積冷凍步驟
mRNA-loaded LNP intermediate 載有 mRNA 的 LNP 中間體
The product consists of an mRNA-lipid complex dispersion that contains the mRNA (CX-024414) that encodes for the pre-fusion stabilised Spike protein of 2019-novel Coronavirus (SARS-CoV-2).Comprehensive development data have been provided. Changes made during development have been described. They included scale-up, addition of a bulk freezing step, change of some equipment and revision of the lipid molar ratios to allow for harmonisation with the platform process.
該產品由一種mRNA-脂質復合分散體組成，其中包含編碼2019年新型冠狀病毒（SARS-CoV-2）融合前穩定 Spike 蛋白的mRNA（CX-024414）。已提供全面的開發數據。已經描述了在開發過程中所做的變更。它們包括放大、增加批量冷凍步驟、改變一些設備和修改脂質摩爾比以與平臺工藝匹配。
Manufacture of the product and process controls
產品生產和工藝控制---涵蓋了冷凍工藝
The LNP manufacturing process comprises lipid stock solution (LSS) preparation, nanoprecipitation mixing, tangential flow filtration (TFF), dilution and cryoprotectant addition, clarification, fill, and freezing and storage.
LNP 制造過程包括脂質儲備溶液 (LSS) 制備、納米沉淀混合、切向流過濾 (TFF)、稀釋和冷凍保護劑添加、澄清、填充以及冷凍和儲存。
Stability of the product
產品的穩定性--現有的凍融工藝對產品的穩定性沒有影響
mRNA-loaded LNP intermediate載有 mRNA 的 LNP 中間體
Stability studies in support of mRNA-loaded LNP intermediate freeze-thaw cycling were performed using the development lot. The results show that the mRNA-1273 LNP biophysical attributes (particle size, polydispersity, encapsulation) are not impacted by the different freeze-thaw processes (room temperature or 2°C to 8°C) or 5 cycles of freeze-thaw
使用開發批次進行了支持負載 mRNA 的 LNP 中間凍融循環的穩定性研究。結果表明，mRNA-1273 LNP 生物物理屬性（粒徑、多分散性、封裝）不受不同凍融過程（室溫或 2°C 至 8°C）或 5 個凍融循環的影響，還進行了支持成品凍融循環的穩定性研究。凍融循環最多重復五次。所有結果都在規格范圍內，表明最終產品在重復凍融步驟方面相對穩定，支持五個凍融循環。然而，批準的產品信息中不允許在上市時反復凍融小瓶。
Finished product
最終成品---仍然需要做凍融測試
Stability studies in support of finished product freeze-thaw cycling were also performed. The freeze-thaw cycling was repeated up to five times. All results were within the specification and indicate that the final product is relatively stable with regard to repeated freeze-thaw steps support five freeze/thaw cycles. Repeated freeze-thaw of vials upon marketing is however, not permitted in the approved product information
Design and conduct of clinical studies
臨床研究的設計和執行---仍需要評估凍融的穩定性，涵蓋運輸和存儲
The RT-PCR assay is acceptably validated and additional validation studies to evaluate stability of clinical samples at various temperatures and freeze/thawing cycles to cover shipping and storage conditions were provided.
RT-PCR 測定得到了可接受的驗證，并提供了額外的驗證研究，以評估臨床樣品在各種溫度和冷凍/解凍循環中的穩定性，以涵蓋運輸和儲存條件。
Why not impacted by the different freeze-thaw processes
為什么凍融工藝對產品沒有影響?
凍融工藝的開發
特殊要求的凍融曲線降低了對mRNA產品的影響
The goal must be to reduce STRESSES DURING FREEZING
目標必須是在凍結過程中減少壓力
Stress is coursed by:
壓力主要這幾方面
• CRYOCONCENTRATION冷凍濃縮
• PH SHIFT pH pH漂移
• PROTEIN INTERACTION WITH THE ICE SURFACE蛋白冰液界面的變性
mRNA Freezing experience
mRNA冷凍實驗
mRNA experienced companies are freezing as fast as possible, so with rates from + to -60° C in < 5hours
經驗豐富的mRNA公司會盡可能快地凍結mRNA（在< 5小時內從+到-60°C凍結）
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