Kelley Brian D, Tannatt Molly, Magnusson Robert, Hagelberg Sigrid, Booth James
Wyeth BioPharma, 1 Burtt Rd., Andover, Massachusetts 01810, USA.
Biotechnol Bioeng. 2004 Aug 5;87(3):400-12. doi: 10.1002/bit.20124.
An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.
开发了一种亲和色谱步骤，用于使用从噬菌体展示文库中选择的肽配体纯化重组B结构域缺失因子VIII（BDDrFVIII）。该肽文库具有多样化的残基，既包含在二硫键约束环内，也包含在环的侧翼。该肽配体在游离溶液中和固定在色谱树脂上时，均以约1 microM的解离常数与BDDrFVIII结合。该肽通过化学合成，亲和树脂通过将该肽偶联到用N-羟基琥珀酰亚胺预活化的琼脂糖基质上制备。优化了偶联条件，以实现一致且完全的配体掺入，并通过测试各种加工极限组合的稳健性研究进行了验证。该肽亲和色谱操作采用的条件与目前用于BDDrFVIII生产的免疫亲和色谱步骤非常相似。该工艺步骤能从复杂的进料流中出色地回收BDDrFVIII，并将宿主细胞蛋白和DNA降低3至4个对数。工艺验证研究表明，树脂可重复使用26个循环，而产品回收率或纯度无变化。进行了一项使用析因设计的稳健性研究，结果表明该步骤对代表商业生产中正常变化的工艺条件的微小变化不敏感。对该工艺步骤进行了缩小模型验证，并用于病毒去除研究。一个涉及浸出肽安全性的验证包包括工艺条件下的浸出率测量、产品池中肽水平的测试、通过加标研究证明下游的稳健去除、终产品测试以及配体的毒理学分析。该肽配体亲和步骤已放大用于BDDrFVIII的cGMP生产，以进行临床试验。
