Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification
Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification
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DOI：
10.1021/bp049840y
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发表时间：
2005-01-01
影响因子：
2.9
通讯作者：
Giardina, SL
中科院分区：
文献类型：
--
作者：
Nellis, DF;Ekstrom, DL;Giardina, SL
描述了生产f5cys - peg -脂质偶联物的gmp合规工艺。这种材料与预先形成的载药脂质体融合，形成“免疫脂质体”，与HER2/neu过表达的癌细胞结合，刺激药物内化，理想地提高了包封药物的治疗指数。通过高密度培养，在大肠杆菌RV308中产生了可溶性单链可变区抗体片段，命名为F5cys。亲和吸附水平翻转流线型rProtein-A树脂强有力地从高压破坏的全细胞匀浆中回收F5cys。鉴定出两种与产品相关的杂质类别：F5cys具有中间序列不连续，F5cys具有pelB先导肽的残留物。低压阳离子交换色谱，在还原条件下在升高的pH下进行，富集了相对于这些杂质的靶F5cys，并制备了用于偶联的c -陆地半胱氨酸。在pH 5.9 +/- 0.1的条件下，通过反应监测和半胱氨酸猝灭，进行定点偶联，得到F5cys-MP-PEG(2000)DSPE。低压粒径排阻色谱法将自发形成的高分子量共轭胶束与低分子量杂质分离。当在10 mM柠檬酸三钠，10%蔗糖（w/v）， pH 6.4（110）中配制1-2 mg/mL时，在-70℃以下保存时，该缀合物是稳定的。比较了六个放大批次。最大的40-L培养产生的F5cys足以制造2,085毫克的结合物，足以支持计划中的临床前和未来的临床试验。经聚丙烯酰胺凝胶电泳测定，该偶联物纯度为93%。杂质主要被确定为与产品有关。残留的内毒素、蛋白A和基因组DNA均处于可接受水平。这项研究成功地解决了扩大免疫脂质体包封疗法的必要步骤。
A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein-A resin robustly recovered F5cys from high-pressure-disrupted, whole-cell homogenates. Two product-related impurity classes were identified: F5cys with mid-sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low-pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C-terrainal cysteine for conjugation. Site-directed conjugation, conducted at pH 5.9 +/- 0.1 with reaction monitoring and cysteine quenching, yielded F5cys-MP-PEG(2000)DSPE. Low-pressure size exclusion chromatography separated spontaneously formed, high-molecular-weight conjugate micelles from low-molecular-weight impurities. When formulated at 1-2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (110), the conjugate was stable when stored below -70 degreesC. Six scale-up lots were compared. The largest 40-L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product-related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale-up of immunoliposome-encapsulated therapeutics.