Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.
Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.
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DOI：
10.1002/bit.27979
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发表时间：
2022-03
影响因子：
3.8
通讯作者：
Love JC
中科院分区：
文献类型：
--
作者：
Dalvie NC;Biedermann AM;Rodriguez-Aponte SA;Naranjo CA;Rao HD;Rajurkar MP;Lothe RR;Shaligram US;Johnston RS;Crowell LE;Castelino S;Tracey MK;Whittaker CA;Love JC
在全球范围内预防COVID-19将需要持续开发大批量、低成本的疫苗生产平台，以满足持续的需求。基于重组蛋白亚基的疫苗候选物仍然很重要，因为它们可以在使用微生物宿主如Komagataella phaffii（巴斯德毕赤酵母）的现有大规模生产设施中以低成本生产。在这里，我们报告了一种改进的和可扩展的SARS-CoV-2刺突蛋白受体结合结构域（RBD）的制造方法;这种蛋白质是几种已报道的候选疫苗的关键抗原。我们通过基因工程改造了一种K. phaffii以满足重组基因对甲醇诱导的需要。无甲醇生产通过减轻蛋白质折叠应激将RBD蛋白的分泌滴度提高> 5倍。从生产工艺中去除甲醇，使规模扩大到1200 L的现有生产设施。这种工程菌株现在用于生产基于RBD的疫苗抗原，目前正在临床试验中，并可用于生产未来疫苗所需的其他RBD变体。作者报告了一种改进的酵母低成本生产工艺，用于临床阶段COVID-19候选疫苗的一种成分。巴斯德毕赤酵母的工程菌株在不使用甲醇的情况下产生抗原，从而改善分泌的蛋白质滴度和细胞健康。取消甲醇作为工艺要求，使技术能够快速转移到现有的大规模生产设施。
Prevention of COVID‐19 on a global scale will require the continued development of high‐volume, low‐cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large‐scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS‐CoV‐2 spike protein receptor‐binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol‐free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre‐existing production facility. This engineered strain is now used to produce an RBD‐based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines. The authors report an improved low‐cost manufacturing process in yeast for a component of a clinical‐stage COVID‐19 vaccine candidate. An engineered strain of Pichia pastoris produces the antigen without using methanol, improving the secreted protein titer and cell health. Elimination of methanol as a process requirement enabled rapid technology transfer to an existing large‐scale production facility.
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影响因子：
5.5
作者：
Guebre-Xabier M;Patel N;Tian JH;Zhou B;Maciejewski S;Lam K;Portnoff AD;Massare MJ;Frieman MB;Piedra PA;Ellingsworth L;Glenn G;Smith G
通讯作者：
Smith G
DOI：
10.1073/pnas.2106845118
发表时间：
2021-09-21
影响因子：
11.1
作者：
Dalvie NC;Rodriguez-Aponte SA;Hartwell BL;Tostanoski LH;Biedermann AM;Crowell LE;Kaur K;Kumru OS;Carter L;Yu J;Chang A;McMahan K;Courant T;Lebas C;Lemnios AA;Rodrigues KA;Silva M;Johnston RS;Naranjo CA;Tracey MK;Brady JR;Whittaker CA;Yun D;Brunette N;Wang JY;Walkey C;Fiala B;Kar S;Porto M;Lok M;Andersen H;Lewis MG;Love KR;Camp DL;Silverman JM;Kleanthous H;Joshi SB;Volkin DB;Dubois PM;Collin N;King NP;Barouch DH;Irvine DJ;Love JC
通讯作者：
Love JC
影响因子：
64.5
作者：
Walls AC;Fiala B;Schäfer A;Wrenn S;Pham MN;Murphy M;Tse LV;Shehata L;O'Connor MA;Chen C;Navarro MJ;Miranda MC;Pettie D;Ravichandran R;Kraft JC;Ogohara C;Palser A;Chalk S;Lee EC;Guerriero K;Kepl E;Chow CM;Sydeman C;Hodge EA;Brown B;Fuller JT;Dinnon KH 3rd;Gralinski LE;Leist SR;Gully KL;Lewis TB;Guttman M;Chu HY;Lee KK;Fuller DH;Baric RS;Kellam P;Carter L;Pepper M;Sheahan TP;Veesler D;King NP
通讯作者：
King NP
影响因子：
3.8
作者：
Vogl, Thomas;Sturmberger, Lukas;Glieder, Anton
通讯作者：
Glieder, Anton
影响因子：
14.9
作者：
McCarthy DJ;Chen Y;Smyth GK
通讯作者：
Smyth GK