Affinity purification and characterization of an anti-PEG IgM.
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.
Pharmaceutical and Analytical R&D, Hoffmann-La Roche, Nutley, NJ 07110, USA. george.ehrlich@roche.com
George K
GK
Ehrlich
Hanspeter
H
Michel
Hitesh P
HP
Chokshi
A Waseem
AW
Malick
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007075:Immunoglobulin M / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000378:metabolism; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D007075:Immunoglobulin M; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C100416:peginterferon alfa-2a