Tom Irene, Lee Vivian, Dumas Michael, Madanat Melanie, Ouyang Jun, Severs Joanne, Andersen John, Buxton Joanne M, Whelan James P, Pan Clark Q
Bayer HealthCare Pharmaceuticals, Biotechnology, Berkeley, CA 94701, USA.
AAPS J. 2007 Jun 22;9(2):E227-34. doi: 10.1208/aapsj0902025.
A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.
一种聚乙二醇化胰高血糖素样肽-1（GLP-1）激动剂和胰高血糖素拮抗剂杂交肽被设计作为2型糖尿病的潜在治疗药物。为支持这种聚乙二醇化双作用糖尿病肽（DAPD）的临床前开发，我们开发了一种可重复的聚乙二醇化、纯化和分析方法。通过评估pH值、反应时间和反应物摩尔比参数，确定了用22 kDa和43 kDa马来酰亚胺-聚乙二醇（马来酰亚胺-PEG）聚合物进行位点特异性聚乙二醇化的最佳条件。开发并成功实施了三步纯化工艺，以纯化聚乙二醇化DAPD并去除过量未偶联的PEG和游离肽。制备了五批起始肽量为100 mg的43 kDa聚乙二醇化DAPD，总产率为53%至71%。通过N端测序、氨基酸分析、基质辅助激光解吸/电离质谱和GLP-1受体激活试验进行的分析表征证实了PEG在位点特异性连接到工程化半胱氨酸残基上，分子量符合预期，氨基酸序列和组成正确，功能活性一致。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、分析离子交换色谱、反相高效液相色谱和鲎试剂检测进行的纯度和安全性分析表明，最终产品中游离肽含量<1%，未偶联PEG含量<5%，每毫克肽内毒素单位<0.2。这些结果表明，我们开发的聚乙二醇化和纯化工艺在以100 mg（基于肽重量）或1.2 g（基于原料药重量）规模生产聚乙二醇化DAPD临床前材料方面是一致且有效的。
