信使RNA（mRNA）是一類(lèi)新興的生物治療藥物。雖然它們有相當(dāng)大的前景，但這一領(lǐng)域仍處于起步階段。mRNA治療為挑戰(zhàn)性疾病的靶向治療和靈活生產(chǎn)提供了新的機(jī)遇，抗COVID-19的mRNA疫苗的快速發(fā)展就證明了這一點(diǎn)。

    mRNA的臨床潛力已經(jīng)超出了疫苗的范疇，擴(kuò)展到其他先進(jìn)的治療方法，例如蛋白質(zhì)替代、基因治療和癌癥免疫治療。

    由于mRNA是脆弱的，不穩(wěn)定的，開(kāi)發(fā) mRNA 疫苗的最重要的挑戰(zhàn)仍然是其固有的不穩(wěn)定性，因?yàn)樗�很有可能在冰點(diǎn)以上分解。

    mRNA 構(gòu)建模塊的修飾以及能夠相對(duì)安全地包裹它顆粒的開(kāi)發(fā)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(chǔ)、運(yùn)輸和管理。

WHO ：
在用 LNP 包封期間以及制造條件下應(yīng)特別注意盡量減少 mRNA 的降解，這個(gè)包封和生產(chǎn)添加會(huì)影響 LNP 及最終 mRNA-LNP 疫苗產(chǎn)品的穩(wěn)定性，例如：產(chǎn)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(jù)某上市的mRNA疫苗的評(píng)估報(bào)告---EMA）
 
Characterisation 特征  ---提供了凍融降解的數(shù)據(jù)
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(qǐng)人提供了mRNA活性物質(zhì)的詳細(xì)特征，包括mRNA CX-024414結(jié)構(gòu)特性的詳細(xì)信息。此外，還提供了色譜圖、熱變性和凍融降解及其對(duì)蛋白質(zhì)表達(dá)的影響數(shù)據(jù)。
 
The composition of the finished product
最終產(chǎn)品的成分---采用蔗糖來(lái)提高凍融和存儲(chǔ)穩(wěn)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(guò)冷凍/解凍和長(zhǎng)期儲(chǔ)存來(lái)提高產(chǎn)品的穩(wěn)定性。
 
Manufacturing process development
生產(chǎn)工藝開(kāi)發(fā)---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(chǎn)品由一種mRNA-脂質(zhì)復(fù)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(wěn)定 Spike 蛋白的mRNA（CX-024414）。已提供全面的開(kāi)發(fā)數(shù)據(jù)。已經(jīng)描述了在開(kāi)發(fā)過(guò)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(shè)備和修改脂質(zhì)摩爾比以與平臺(tái)工藝匹配。
 
Manufacture of the product and process controls
產(chǎn)品生產(chǎn)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(guò)程包括脂質(zhì)儲(chǔ)備溶液 (LSS) 制備、納米沉淀混合、切向流過(guò)濾 (TFF)、稀釋和冷凍保護(hù)劑添加、澄清、填充以及冷凍和儲(chǔ)存。
 
Stability of the product
產(chǎn)品的穩(wěn)定性--現(xiàn)有的凍融工藝對(duì)產(chǎn)品的穩(wěn)定性沒(méi)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(kāi)發(fā)批次進(jìn)行了支持負(fù)載 mRNA 的 LNP 中間凍融循環(huán)的穩(wěn)定性研究。結(jié)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(guò)程（室溫或 2°C 至 8°C）或 5 個(gè)凍融循環(huán)的影響，還進(jìn)行了支持成品凍融循環(huán)的穩(wěn)定性研究。凍融循環(huán)最多重復(fù)五次。所有結(jié)果都在規(guī)格范圍內(nèi)，表明最終產(chǎn)品在重復(fù)凍融步驟方面相對(duì)穩(wěn)定，支持五個(gè)凍融循環(huán)。然而，批準(zhǔn)的產(chǎn)品信息中不允許在上市時(shí)反復(fù)凍融小瓶。
 
Finished product
最終成品---仍然需要做凍融測(cè)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(shè)計(jì)和執(zhí)行---仍需要評(píng)估凍融的穩(wěn)定性，涵蓋運(yùn)輸和存儲(chǔ)
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(cè)定得到了可接受的驗(yàn)證，并提供了額外的驗(yàn)證研究，以評(píng)估臨床樣品在各種溫度和冷凍/解凍循環(huán)中的穩(wěn)定性，以涵蓋運(yùn)輸和儲(chǔ)存條件。
 
Why not impacted by the different freeze-thaw processes
為什么凍融工藝對(duì)產(chǎn)品沒(méi)有影響?
凍融工藝的開(kāi)發(fā)
特殊要求的凍融曲線(xiàn)降低了對(duì)mRNA產(chǎn)品的影響
The goal must be to reduce STRESSES DURING FREEZING
目標(biāo)必須是在凍結(jié)過(guò)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(shí)驗(yàn)
mRNA experienced companies are freezing as fast as possible, so with rates from + to -60° C in < 5hours
經(jīng)驗(yàn)豐富的mRNA公司會(huì)盡可能快地凍結(jié)mRNA（在< 5小時(shí)內(nèi)從+到-60°C凍結(jié)）