Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation.
Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation.
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DOI：
10.1371/journal.pone.0164053
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Remarque EJ
中科院分区：
文献类型：
--
作者：
Faber BW;Hellwig S;Houard S;Havelange N;Drossard J;Mertens H;Croon A;Kastilan R;Byrne R;van der Werff N;van der Eijk M;Thomas AW;Kocken CH;Remarque EJ
恶性疟原虫顶端膜抗原1（PfAMA 1）是用于疟疾的主要无性生殖血液阶段疫苗候选物。在临床试验的准备中，三种多样性覆盖（DiCo）PfAMA 1胞外域蛋白，旨在克服PfAMA 1中存在的内在多态性，在巴斯德毕赤酵母中按照良好生产规范（GMP）生产。使用相同的方法，在70-L规模的补料分批发酵中培养3种菌株，并通过两个色谱步骤（超滤/渗滤程序和尺寸排阻色谱）纯化PfAMA 1-DiCos，得到高纯度（>95%）的PfAMA 1-DiCo 1、PfAMA 1 DiCo 2和PfAMA 1 DiCo 3，最终产量分别为1.8、1.9和1.3克。根据SDS-PAGE（2条主带）和MS数据，N-末端测定显示，每种蛋白质的约50%从其N-末端丢失12个残基。在还原条件下，在二硫键结合区域内的有限蛋白水解裂解位点变得明显。这三种蛋白质定量结合到识别构象表位的mAb 4G 2，表明蛋白质的正确折叠。冻干制剂（PfAMA 1-DiCo 1、DiCo 2、DiCo 3的1：1：1混合物）符合所有预设放行标准（外观、溶出速率、鉴别、纯度、蛋白质含量、水分含量、不溶性微粒、免疫效力（用佐剂复溶后）、异常毒性、无菌性和内毒素），在-20 ℃加速和实时稳定性研究中稳定超过24个月。当与选择用于临床I期评价的佐剂一起配制时，制剂在家兔重复给药毒性研究中未显示不良反应。该制剂已进入Ia/Ib期临床试验。
Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.
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