Ehrlich George K, Michel Hanspeter, Chokshi Hitesh P, Malick A Waseem
Pharmaceutical and Analytical R&D, Hoffmann-La Roche, Nutley, NJ 07110, USA.
J Mol Recognit. 2009 Mar-Apr;22(2):99-103. doi: 10.1002/jmr.920.
Anti-PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti-PEG IgM was observed using the 30 kDa PEG-derivatized NuGel (single passage). Purified anti-PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti-PEG IgM, in solution and adsorbed on 20 kDa PEG-derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS-PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution-digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N-terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti-PEG IgM fragments were characterized for binding to pegylated interferon alfa-2a by ELISA. The results from these studies suggest that affinity purified anti-PEG IgM and fragments can be used as probes in detection assays for PEG functionalized biotherapeutics in pre-clinical and clinical studies.
使用可变长度的聚乙二醇链（5、10、20和30 kDa）作为亲和配体，通过亲和色谱法纯化抗聚乙二醇IgM。使用30 kDa聚乙二醇衍生化的NuGel（单次通过）观察到抗聚乙二醇IgM的最大结合。通过表面等离子体共振、蛋白质免疫印迹和酶联免疫吸附测定法对纯化的抗聚乙二醇IgM与聚乙二醇功能化的蛋白质/肽的结合进行表征。将溶液中的抗聚乙二醇IgM和吸附在20 kDa聚乙二醇衍生化的NuGel上的抗聚乙二醇IgM进行胃蛋白酶消化，然后进行亲和色谱法。两种制剂洗脱液的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析产生了一个大小相似的片段。然而，在溶液消化的亲和纯化材料的洗脱液中观察到一条额外的低分子量条带，而在亲和基质上进行胃蛋白酶消化的材料的洗脱液中不存在该条带。较低分子量的片段可以在较温和的条件下洗脱，表明结合多样性的丧失。质谱分析得出相应片段的分子量分别为132 kDa（两者）和82 kDa（溶液）。两个片段的N端测序产生的一级序列（重链和轻链）不仅彼此相同，而且与天然IgM的序列相同。通过酶联免疫吸附测定法对抗聚乙二醇IgM片段与聚乙二醇化干扰素α-2a的结合进行表征。这些研究结果表明，亲和纯化的抗聚乙二醇IgM及其片段可作为探针，用于临床前和临床研究中聚乙二醇功能化生物治疗药物的检测测定。
